Expression vectors comprising nucleic acids encoding SEBF proteins and uses thereof

ABSTRACT

The invention relates to vectors comprising nucleic acid sequences encoding transcriptional repressor silencing element binding factor referred as “SEBF”, transformed vegetal host and plants comprising said vectors. The invention also relates to methods of altering resistance of a plant to pathogens with SEBF encoding nucleic acids.

RELATED APPLICATION

This application claims priority of U.S. Provisional Application60/303,780 filed Jul. 10, 2001, the disclosure of which is incorporatedby reference herein in its entirety.

BACKGROUND OF THE INVENTION

A) Field of the Invention

The present invention relates to a plant gene regulatory element and itsuses, and more particularly to a silencing element for modulating plantresponses to pathogens, auxin and ethylene. The invention also relatesto transcriptional repressors which specifically binds onto thesilencing element of the invention, including a protein referred hereinafter as “SEBF”.

B) Brief Description of the Prior Art

A variety of defense specific events are induced in plants in responseto pathogen infection. Although key components of the signaling cascadesare being discovered, few transcription factors that integrate thesesignals at the transcriptional level have been identified up to date.

PR genes are plant genes that are induced by pathogen invasion. Thesegenes are subdivided into 11 classes. Since PR genes are wellcharacterized, they provide excellent models to study transcriptionalregulation of defense genes.

The PR-10 gene family is one of the classes of PR genes. Expressionstudies have identified cis-acting elements involved in PR-10a generegulation, a member of the PR-10 gene family. An elicitor responseelement (ERE) located between nucleotides −135 and −105 is essential andsufficient for elicitor induced expression of PR-10a. PBF-2, asingle-stranded DNA binding factor, appears to play a role in activationof PR-10a from the ERE. It has been shown that the presence of the EREis sufficient for PR-10a activation, removal of the silencing element(SE), located between −52 and −27, leads to further activation,suggesting that SE participates, with the ERE, in the regulation ofPR-10a (Matton et al, 1993; Després et al., 1995). However, the exactnucleic acid sequence required for full SE activity has never beengiven. Furthermore, the identity of the transcriptional repressorspecifically binding to the silencing element (SE) of PR-10a is alsounknown.

Accordingly, there is a need for an isolated or purified nucleic acidcomprising a sequence coding for full of SE activity and to the usethereof for modulating activity of genes, and more particularly genesinvolved in plant responses to pathogen such as PR-10a gene. There isalso a need for methods and genetically modified plants wherein thenucleic acid of the invention has been introduced or wherein thesequence coding for full SE activity has been mutated, deleted, orsilenced, thereby modulating the plant defense mechanisms and resistanceto pathogens.

There is also a long felt need for a transcriptional repressor that iscapable of modulating plant defense mechanisms and resistance topathogens and more particularly for a transcriptional repressor capableto specifically bind the isolated or purified nucleic acid of theinvention. There is also a need for methods and genetically modifiedplants wherein levels of the transcriptional repressor of the inventionhave been modulated.

There is a further need for effective methods and compositions tomodulate plant resistance or tolerance to pathogens, and/or to modulateplant response to auxin and/or to ethylene.

The present invention fulfills these needs and also other needs whichwill be apparent to those skilled in the art upon reading the followingspecification.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention relates to anisolated or purified nucleic acid molecule comprising a sequenceselected from the group consisting of:

-   a) sequence set forth in SEQ ID NO: 21;-   b) a nucleotide sequence having at least 96% nucleotide sequence    identity with SEQ ID NO: 21; and-   c) a nucleotide sequence having at least 75% nucleotide sequence    identity with a nucleic acid encoding an amino acid sequence of SEQ    ID NO:22.

The invention also concerns transformed or transfected cells as well ascloning or expression vector that contains such a nucleic acid.Preferably, the cell and the vector express or are capable of directingexpression of the peptide encoded by the nucleic acid.

In a related aspect, the invention concerns an isolated or purifiedprotein comprising an amino acid sequence selected from the groupconsisting of:

-   a) sequences encoded by a nucleic acid as defined previously;-   b) sequences having at least 85% identity to SEQ ID NO: 22;-   c) sequences having at least 87% similarity to SEQ ID NO: 22;-   d) sequence set forth in SEQ ID NO: 22;-   e) sequences having at least 85% identity to amino acid sequences    encoded by nucleotides 68 to 937 of SEQ ID NO: 21; and-   f) sequences having at least 87% sequence similarity to amino acid    sequences encoded by nucleotides 68 to 937 of SEQ ID NO: 21.

The inventions also concerns compositions comprising any of the nucleicacids or protein defined previously.

In another aspect, the invention relates to plant proteinic nuclearfactors that are capable, among other things, of mediating repression ofa silencing element involves in plant defense mechanisms. Preferably,the proteinic nuclear factor is a plant transcriptional repressor whichspecifically binds onto the sequence BTGTCNC or YTGTCNC. Most preferredtranscriptional repressor consists of a protein referred herein after as“SEBF” for Silencing Element Binding Factor. In one embodiment there isdescribed a composition comprising an isolated or purified SEBF proteinor a functional homologue thereof. Preferably, the SEBF protein orhomologue comprises an amino acid sequence selected from the groupconsisting of:

-   a) sequences encoded by a nucleic acid having a sequence at least    85% identical to nucleotides 68 to 937 of SEQ ID NO: 21;-   b) sequences having at least 85% identity to SEQ ID NO:22;-   c) sequences having at least 87% similarity to SEQ ID NO:22; and-   d) sequence provided in SEQ ID NO:2.

More preferably, the SEBF protein is purified from potato, and whereinit has a purification factor of about 90 to about 20 700 fold.

According to another aspect, the present invention relates to anisolated or purified nucleic acid comprising a binding sequence ontowhich proteinic nuclear factors, such as the transcriptional repressorof the silencing element (SE) of PR-10a, specifically binds. Preferably,the binding sequence comprises sequence BTGTCNC (SEQ ID NO:23), morepreferably sequence YTGTCNC (SEQ ID NO:24). The invention also concernsan isolated or purified gene regulatory element comprising a nucleicacid sequence that is essential for the full activity in plant of thesilencing element (SE) of PR-10a. Preferably, the gene regulatoryelement consists of a silencing element and it comprises sequenceGACTGTCAC (SEQ ID NO:26) or sequence BTGTCNC (SEQ ID NO:23), and morepreferably sequence YTGTCNC (SEQ ID NO:24). The invention also concernsa DNA construct comprising the gene regulatory element and geneticallymodified plant entities comprising the gene regulatory element or theDNA construct.

According to a related aspect, the present invention concerns a methodfor altering gene expression in a plant. The method comprises the stepof altering in the plant binding of a nuclear DNA-binding protein tosequence BTGTCNC. A non-limitative list of preferred endogenousDNA-binding proteins includes those having at least 48% identity orsimilarity to SEBF. More preferably, the DNA-binding protein consists ofSEBF or of a functional homologue thereof having at least 90% identityor similarity to SEBF.

In a preferred embodiment, there is described a method for increasingthe expression of a gene of interest, this gene having a promoter regioncomprising sequence BTGTCNC. The method comprises the step of mutatingthe promoter region of the gene for mutating or deleting the sequenceBTGTCNC. The gene maybe PR gene.

In another preferred embodiment, there is described a method forreducing the expression of a gene of interest, this gene having apromoter region devoid of sequence BTGTCNC. The method comprises thestep of introducing in an operable linked manner the sequence BTGTCNCinto the promoter region.

In a more specific embodiment, there is described a vegetal host (e.g.algae, plant) genetically modified for exhibiting an altered expressionor biological activity of a proteinic nuclear factor having a specificbinding activity to sequence BTGTCNC (SEQ ID NO:23), preferably YTGTCNC(SEQ ID NO:24), the altered level being compared to a correspondinggenetically unmodified vegetal host in which the endogenous level hasnot been altered. Preferred proteinic nuclear factors are those havingat least 48% identity or similarity to SEBF. More preferably, theproteinic nuclear factors consists of SEBF or of a functional homologuethereof having at least 90% identity or similarity to SEBF.

In an even more specific embodiment, the expression or biologicalactivity of SEBF or homologue has been increased in the vegetal hostsuch that it exhibits a phenotype selected from the group consisting of:

-   reduced resistance or tolerance to a pathogen;-   reduced growth, rooting and/or fruit production;-   increased resistance to an auxinic herbicide;-   reduced ethylene production;-   delay in ripening of its fruit(s) and/or protection of its fruit(s)    against over-ripening.

In an other specific embodiment, the expression or biological activityof SEBF or homologue has been reduced in the vegetal host such that itexhibits a phenotype selected from the group consisting of

-   increased resistance or tolerance to a pathogen;-   increased growth, rooting and/or fruit production;-   increased sensitivity to an auxinic herbicide;-   increased ethylene production; and-   early fruit maturation.

The invention also encompasses methods for obtaining the vegetal hosthaving the phenotype(s) described previously. Typically these methodscomprises the step of modulating (typically reducing or increasing) inthe plant expression or biological activity of SEBF or of a SEBFfunctional homologue.

Another related aspect of the invention concerns a genetically modifiedvegetal host comprising a genome, wherein transcriptional activity of agene associated with presence or absence of sequence BTGTCNC (SEQ IDNO:23), preferably YTGTCNC (SEQ ID NO:24), in a promoter region thisgene has been altered. Of course, the altered biological activity iscompared to a corresponding genetically unmodified vegetal host in whichthe endogenous biological activity has not been altered.

In one embodiment, the promoter region of the gene comprises thesequence BTGTCNC, and the promoter region has been genetically modified(e.g. mutation, deletion) for inactivating a repressive transcriptionalactivity associated with the presence of the sequence BTGTCNC.

In another embodiment, the promoter region is devoid of sequence BTGTCNC(SEQ ID NO:23), and this region has been genetically modified forinserting therein in an operable linked manner the sequence BTGTCNC.

In another aspect, the present invention relates to methods forobtaining a particular phenotype in plants, and more particularly plantswhich are used in agriculture and plants with a horticultural value. Inone embodiment, there is described methods for:

-   the modulation of a plant resistance or tolerance to pathogens;-   the modulation of induction of genes of a plant controlled by    auxins;-   the modulation of a plant auxins-controlled genes induction;-   the augmentation of growth, rooting and/or fruit production in a    plant;-   the modulation of a plant auxin-inducted ACC synthase gene;-   the modulation of a plant ethylene production; and-   the modulation of a plant plastid mRNAs stability, expression or    activity.

All these methods comprises the step of modulating in the plantexpression or biological activity of an endogenous SEBF protein or of aSEBF functional homologue.

In one preferred embodiment, there is described a method for obtaining agenetically modified plant exhibiting a phenotype selected from thegroup consisting of:

-   increased resistance or tolerance to a pathogen;-   increased growth, rooting and/or fruit production;-   increased sensitivity to an auxinic herbicide;-   increased ethylene production;-   early fruit maturation;-   altered plastid mRNAs stability, expression or activity;-   a promoter region of a gene involved in the phenotype comprising    sequence BTGTCNC (SEQ ID NO:23), the phenotype of the plant being    compared to a corresponding genetically unmodified plant;-   the method comprising the step of genetically modifying the genome    of this plant for inactivating an endogenous biological activity    associated with the presence of the sequence BTGTCNC.

In one preferred embodiment, there is described a method for obtaining agenetically modified plant exhibiting a phenotype selected from thegroup consisting of:

-   reduced resistance or tolerance to a pathogen;-   reduced growth, rooting and/or fruit production;-   increased resistance to an auxinic herbicide;-   reduced ethylene production;-   delay in ripening of its fruit(s) and/or protection of its fruit(s)    against over-ripening; and-   altered plastid mRNAs stability, expression or activity;-   a promoter region of a gene involved in the phenotype being devoid    of sequence BTGTCNC (SEQ ID NO:23), the phenotype of the plant being    compared to a corresponding genetically unmodified plant;-   the method comprising the step of genetically modifying the genome    of said plant for inserting therein in an operably linked manner the    sequence BTGTCNC.

In further embodiments of the invention, there is described plants, andmethods for obtaining the same, the plants exhibiting an increased (ordecreased) resistance or tolerance to pathogens; a faster (or lower)induction of its defense response; an increased (or decreased)sensitivity for endogenous auxins; and/or a delayed ripening or anadvanced fruit maturation.

In one specific embodiment, there is provided a method for modulating aplant resistance or tolerance to a pathogen, comprising modulating inthe plant expression or biological activity of an endogenous SEBFprotein. More particularly, the is described a method for increasing aplant resistance or tolerance to a pathogen, comprising reducing in theplant expression or biological activity of an endogenous SEBF protein.Expression or biological activity of the endogenous SEBF protein may bereduced increased for instance by expressing in the plant SEBF antisensemolecules; by expressing proteins inducing a co-suppression of SEBFlevel or activity; by a knock out or a chemical mutagenesis of a geneencoding the SEBF protein; of by expressing a ribozyme cleaving a SEBFmRNA. The is also described a method for reducing a plant resistance ortolerance to a pathogen, comprising increasing in the plant expressionor biological activity of a SEBF protein. The expression or biologicalactivity of the endogenous SEBF protein may increased for instance byintroducing in the plant a expressible SEBF coding sequence. The SEBFcoding sequence may be under control of an inducible or constitutivepromoter. The invention also encompasses plants genetically modified forhaving an increased (or reduced) resistance or tolerance to a pathogenwhen compared to a corresponding plant not genetically modified, whereinexpression or biological activity of an endogenous SEBF protein isreduced (or increased) in the genetically modified plant as compared toa corresponding genetically unmodified plant.

In a another specific embodiment, there is provided a method formodulating a plant resistance or tolerance to a pathogen, the planthaving sequence BTGTCNC (SEQ ID NO:23) in a promoter region of a gene,the method comprising altering in this plant the binding of anendogenous nuclear DNA-binding protein to the sequence BTGTCNC. Moreparticularly, the is described a method for increasing a plantresistance or tolerance to a pathogen, the plant having sequence BTGTCNC(SEQ ID NO:23) in a promoter region of a gene, the method comprisingreducing or preventing in the plant binding of an endogenous nuclearDNA-binding protein to the sequence BTGTCNC. The is also described amethod for reducing a plant resistance or tolerance to a pathogen,comprising permitting or increasing in said plant binding of anendogenous nuclear DNA-binding protein to a promoter region of a geneincluding sequence BTGTCNC (SEQ ID NO:23). The invention alsoencompasses plants genetically modified by these methods for having anincreased (or reduced) resistance or tolerance to a pathogen.

In a further specific embodiment, there is provided a method formodulating induction of genes controlled by auxins in plants, the methodcomprising modulating in the plant expression or biological activity ofan endogenous SEBF protein or of a SEBF functional homologue. Moreparticularly, the is described a method for increasing growth, rootingand/or fruit production in a plant, the method comprising reducing inthe plant expression or biological activity of an endogenous SEBFprotein or of a SEBF functional homologue. There is also described amethod for increasing a plant resistance to an auxinic herbicide,comprising reducing in the plant expression or biological activity of anendogenous SEBF protein or of a SEBF functional homologue. The inventionalso encompasses plants genetically modified by these methods.

In still a further specific embodiment, there is provided a method formodulating a plant ethylene production, comprising modulating in theplant expression or biological activity of an endogenous SEBF protein orof a SEBF functional homologue. More particularly, the is described amethod for increasing production of ethylene by a plant, comprisingreducing in the plant expression or biological activity of an endogenousSEBF protein or of a SEBF functional homologue. There is also describeda method for reducing production of ethylene by a plant, comprisingincreasing in the plant expression or biological activity of anendogenous SEBF protein or of a SEBF functional homologue. The inventionalso encompasses plants genetically modified by these methods for havingan increased (or reduced) production of ethylene.

As used hereinbefore, the vegetal host preferably consists of a plant(monocotyledon or dicotyledon), more preferably a vegetable, aleguminous plant, a tree, a fruit tree, grass, a cereal, and even morepreferably it consists of a potato, a tomato, tobacco, cotton, rice,wheat, corn, barley, oat, canola, soybean, pea, sugar cane, sugar beet,strawberry, and banana.

Other objects and advantages of the present invention will be apparentupon reading the following non-restrictive description of severalpreferred embodiments, made with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a picture of a gel showing binding of SEBF to asingle-stranded silencing element. EMSA was performed with 10 μg ofcrude nuclear preparation and 20,000 CPM of ³²P labeled SE coding strand(lane 1; CS) or non-coding strand (lane 3; NCS). The double-strandedprobe (DS) was made by annealing radiolabeled non-coding and non-labeled(cold) coding strand. The ratio CS:NCS is 0.75:1, 1.5:1 and 3:1 forlanes 4 through 6, respectively. No extract was added in lane 2. Arrowsindicate the position of the CS, NCS and DS probes and of the SEBF shiftin the gel.

FIGS. 2A, 2B, 2C and 2D show mutational analysis of potato silencingelement (SE). FIG. 2A: Schematic representation of the reporterconstructs and mutant oligonucleotides used in this study. Reporterconstructs contain the PR-10a promoter region from −135 to +136 fused tothe bacterial gene uidA encoding β-glucuronidase (GUS). The position ofthe elicitor response element (ERE), the silencing element (SE) and theTATA box are shown (TATA). The transcriptional start site is indicatedby an arrow. The common sequence between the wild-type (WT; SEQ IDNO:45) and mutant oligonucleotides m1 to m5 (SEQ ID NOs:1 to 5) isindicated by dashed lines, and mutated nucleotides are represented bylowercase letters. FIG. 2B: EMSA studies using 10 μg of crude potatotuber nuclear preparation and 20,000 CPM of the ³²P labeledsingle-stranded CS oligonucleotides presented in FIG. 2A. FIG. 2C: EMSAstudies using 10 ng of purified recombinant SEBF and 20,000 CPM of the³²P labeled single-stranded CS oligonucleotides presented in FIG. 2A.FIG. 2D: The sequence from −52 to −27 of the PR-10a promoter fused tothe uidA gene was replaced by the sequences presented in FIG. 2A. Theresulting plasmids were electroporated in potato leaf protoplasts andthe GUS activity was measured. The histogram represents fold activity towild type (WT=1). Transfection efficiencies were corrected byco-electroporating a luciferase reporter gene. Results represent themean from a minimum of 6 individual electroporations. Error barsindicate the standard deviation of the mean.

FIGS. 3A, 3B and 3C show results leading to the determination of theconsensus SEBF binding site. FIG. 3A: Oligonucleotides used for finemapping of the SEBF binding site are listed. The common sequence betweenwild-type (SEQ ID NO:46) and mutant oligonucleotides m6 to m20 (SEQ IDNOS:6 to 20) is indicated by dashed lines. Mutated nucleotides arerepresented by lowercase letters. FIG. 3B: EMSA showing the binding ofSEBF to oligonucleotides containing two mutated nucleotides. FIG. 3C: Asin FIG. 3B except oligonucleotides containing single mutation. Studieswere performed using 10 μg of crude nuclear preparation and 20,000 CPMof ³²P labeled single-stranded oligonucleotides presented in FIG. 3A.

FIG. 4 is a picture of a gel showing the purification of SEBF. Coomassiestaining of proteins from each step of the purification of SEBF (lanes 1through 4). The crude nuclear extract (Crude; lane 1) was loaded on aQ-Sepharose™ column. SEBF was eluted at 400 mM NaCl (Q-seph; lane 2)before two rounds of affinity purification (Aff.1, Aff.2; lane 3, lane4). Lane 5 shows a southwestern (SW) experiment done with affinity 2purified SEBF and the wild type coding strand as a radiolabeled probe.Arrows indicate the two purified bands. Molecular weight markers areindicated on the left.

FIG. 5 shows the amino acid sequence of SEBF (SEQ ID NO:22; GenBank™accession No AF38431). The predicted site for transit peptide cleavageis indicated by an arrow. The two consensus sequence type RNA bindingdomains (cs-RBD) are underlined. The amino acid sequence obtainedthrough amino terminal sequencing is shown in bold characters.

FIG. 6 shows cellular localization of SEBF. Potato leaves werefractionated into cytoplasm, nuclei and chloroplasts. Ten μg of eachfraction were separated by 12% SDS-PAGE. Proteins were transferred tonitrocellulose and the presence of SEBF was revealed using an anti-SEBFantibody. The first lane (10 ng of recombinant SEBF) shows the proteinwhich served to immunize the rabbits. Chlorophyll (CHL) was used as achloroplastic marker (data is presented as μg of chlorophyll [mg ofprotein]⁻¹) and alcohol dehydrogenase (ADH) as a cytoplasmic marker(data is presented as increased OD [mg of protein]⁻¹[min]⁻¹). N.D., notdetectable.

FIGS. 7A, 7B and 7G show the genomic organization of SEBF. FIG. 7A:Southern blot of SEBF. Five μg of digested genomic DNA was loaded perlane and probed with a random-labeled Xmnl fragment from the SEBF cDNAshown in FIG. 7C. Molecular markers are indicated on the right. FIG. 7B:PCR analysis of the genomic DNA. The position of the oligonucleotides (1to Z; lane 1 to 6) on the cDNA is presented in FIG. 7C. The differencein size between the amplification products of lanes 5 and 6, lanes 3 and4, and lanes 2 and 4, define the size of introns 1, 2 and 3respectively. Molecular weight markers are indicated on the right. FIG.7C: Deduced genomic organization of SEBF. The cDNA is represented as aline and the coding region as a box. The amino terminal transit sequenceis presented in black. Introns are indicated as triangles emerging fromthe cDNA. The oligonucleotides used in the PCR reactions are designatedby letters (T, U, V, W, X, Y, Z; corresponding to SEQ ID NOS: 38 to 44).The position and size of the introns are deduced from the PCR analysisFIG. 7B. Restriction sites are indicated by arrows: HinDIII (H), EcoRI(E), Xmnl (Xn).

FIG. 8 shows that SEBF overexpression represses PR-10a expression. Thecoding sequence of precursor SEBF (containing the putative transitpeptide) and the coding sequence of a control protein were each insertedinto plasmid pBI223D (Effector Plasmids). These plasmids wereco-electroporated in potato leaf protoplasts with the reporter plasmidsdescribed in FIG. 2D (Reporter Plasmids). The histogram represents theeffect of SEBF overexpression on reporter activity compared to theoverexpression of the control protein (control=100). Fold activity towild type SE (WT=1) is presented for easier reference to FIG. 2D.Transfection efficiencies were corrected by co-electroporating aluciferase reporter gene. Results represent the mean from a minimum of 3individual electroporations. Error bars indicate the standard deviationof the mean.

FIGS. 9A and 9 b show that SEBF binds the promoter of other defensegenes. FIG. 9A: Oligonucleotides used in this experiment are listed.Nucleotides diverging from PR-10a (SEQ ID NO: 25 and 47; correspondingto nucleotides 1426 to 1450 of Genbank™ acc. No. M29041) are representedby lowercase letters. The SEBF binding site is underlined. ChtC2 is achitinase gene from potato (SEQ ID NO:48, corresponding to nucleotides1264 to 1288 of Genbank™ acc. No. AF153195) and CHN50 is a chitinasegene from tobacco (SEQ ID NO:49, corresponding to nucleotides 1215 to1239 of Genbank™acc. No. X51599). FIG. 9B: EMSA studies using 0.5 μg of400 mM Q-Sepharose fraction and 20,000 CPM of the ³²P labeledsingle-stranded oligonucleotides presented in FIG. 9A.

FIG. 10 shows the DNA (SEQ ID NO:21) and protein (SEQ ID NO:22) sequenceof SEBF (GenBank™ acc. No. AF38431). The translated protein (see FIG. 5;SEQ ID NO:22) is indicated below the DNA sequence. Each amino acid isindicated below the first nucleotide of the codon.

FIGS. 11A, 11B and 11C show the binding of SEBF to the AuxRE sequencepresent in the GH3 promoter of soybean. FIG. 11A: Comparison of the SEBFbinding site (SEQ ID NO:23) with the AuxRE (SEQ ID NO:55). FIG. 11B:Oligonucleotides used in this study (SEQ ID NOS:50 and 51). FIG. 11C:EMSA was performed with 0.5 μg of a 400 mM Q-sepharose™ fraction and20,000 CPM of ³²P labeled oligonucleotides presented in FIG. 11B.

FIGS. 12A, 12B and 12C show the functionality of the SE sequence in thepromoter of the defense gene CHN50. FIG. 12A: EMSA was performed with0.5 μg of a 400 mM Q-sepharose™ fraction and 20,000 CPM of ³²P labeledoligonucleotides presented in FIG. 12C. FIG. 12B: The CHN50 promoter, ormutated versions shown in FIG. 12C, were fused to the uidA gene. Theresulting plasmids were electroporated in tobacco leaf protoplasts andthe GUS activity was measured. Transformation efficiencies werecorrected by co-electroporating a luciferase reporter gene. Resultsrepresent a minimum of three individual electroporations. Error barsindicate the standard deviation of the mean. FIG. 12C: oligonucleotidesused in this study (WT=SEQ ID NO:52; M1=SEQ ID NO:53; M2=SEQ ID NO:54).Mutations are represented by lowercase letters and are underlined. TheSEBF binding site is indicated in boldface characters.

DETAILED DESCRIPTION OF THE INVENTION

A) Definitions

In order to provide an even clearer and more consistent understanding ofthe specification and the claims, including the scope given herein tosuch terms, the following definitions are provided:

Antisense: Refers to nucleic acids molecules capable of regulating theexpression of a corresponding gene in a plant. An antisense molecule asused herein may also encompass a gene construct comprising a structuralgenomic gene, a cDNA gene or part thereof in reverse orientationrelative to its or another promoter. Typically antisense nucleic acidsequences are not template for protein synthesis but yet interact withcomplementary sequences in other molecules (such as a gene or RNA)thereby causing the function of those molecules to be affected.

Chemical derivative: As used herein, a protein/polypeptide is said to bea “chemical derivative” of another protein/polypeptide when it containsadditional chemical moieties not normally part of the protein/peptide,said moieties being added by using techniques well known in the art.Such moieties may improve the protein/polypeptide solubility,absorption, bioavailability, biological half life, and the like. Anyundesirable toxicity and side-effects of the protein/peptide may beattenuated and even eliminated by using such moieties. For example,proteins/polypeptides can be covalently coupled to biocompatiblepolymers (polyvinyl-alcohol, polyethylene-glycol, etc) in order toimprove stability or to decrease/increase their antigenicity.

Defence gene: A gene that is induced and/or involved in a plant responseto a pathogen challenge.

Fragment: refers to a section of a molecule, such as protein/polypeptideor nucleic acid, and is meant to refer to any portion of the amino acidor nucleotide sequence.

Functional homologue: As is generally understood and used herein, refersto non-native a polypeptide or a nucleic acid molecule that possesses afunctional biological activity that is substantially similar to thebiological activity of a native polypeptide or a nucleic acid molecule.Preferred functional homologue are polypeptides or nucleic acidmolecules having a sequence “substantially identical” (see hereinafter)to the native polypeptide or a nucleic acid molecule. The functionalhomologue may exist naturally or may be obtained following a single ormultiple amino acid substitutions, deletions and/or additions relativeto the naturally occurring enzyme(s) using methods and principles wellknown in the art. A functional homologue of a protein may or may notcontain post-translational modifications such as covalently linkedcarbohydrate, if such modification is not necessary for the performanceof a specific function. It should be noted, however, that nucleotide oramino acid sequences may have similarities below the above givenpercentages and still encode a proteinic molecule having a desiredactivity, and such proteinic molecules may still be considered withinthe scope of the present Invention where they have regions of sequenceconservation. The term “functional homologue” is intended to the“fragments”, “segments”, “variants”, “analogs” or “chemical derivatives”of a polypeptide or a nucleic acid molecule.

Fusion protein: A protein formed by the expression of a hybrid gene madeby combining two gene sequences. Typically, this is accomplished bycloning a cDNA into an expression vector in frame with an existing gene.

Genetic modification: When used with the term “vegetal host” or “plant”it refers to the introduction of an exogenous nucleic acid into one ormore vegetal host (plant) cells to create a genetically modified vegetalhost or plant. Methods for genetically modifying vegetal host such asplants are well known in the art. In some cases, in may be preferablethat the genetic modification is permanent such that the geneticallymodified plant may regenerate into whole, sexually competent, viablegenetically modified plants. A plant genetically modified in a permanentmanner would preferably be capable of self-pollination orcross-pollination with other plants of the same species, so that theexogenous nucleic acid, carried in the germ line, may be inserted intoor bred into agriculturally useful plant varieties.

Endogenous or Endogenous level(s): Refers to a given substance or to theconcentration of a given substance which is normally found in a plant(intrinsic) at a given time and stage of growth. The term also includesfunctional homologues of a given substance or protein which may resultsfrom a mutation. Reference herein is made to the altering of theendogenous level of a compound or of an enzyme activity relating to anelevation or reduction in the compound's level or enzyme activity of upto 30% or more preferably of 30, 35, 40, 45 or 50%, or even morepreferably 55, 60, 65, 70 or 75% or still more preferably 80, 85, 90,95% or greater above or below the normal endogenous or existing levels.The levels of a compound or the levels of activity of an enzyme can beassayed using known method and techniques.

Expression: refers to the process by which gene encoded information isconverted into the structures present and operating in the cell. In thecase of cDNAs, cDNA fragments and genomic DNA fragments, the transcribednucleic acid is subsequently translated into a peptide or a protein inorder to carry out its function if any. The term “overexpression” refersto an upward deviation respectively in assayed levels of expression ascompared to a baseline expression level which is the level of expressionthat is found under normal conditions and normal level of functioning.Similarly, the term “underpression” refers to an downward deviation. By“positioned for expression” is meant that the DNA molecule is positionedadjacent to a DNA sequence which directs transcription and translationof the sequence (i.e., facilitates the production of, e.g., a NAIPpolypeptide, a recombinant protein or a RNA molecule).

Isolated or Purified or Substantially pure: Means altered “by the handof man” from its natural state, i.e., if it occurs in nature, it hasbeen changed or removed from its original environment, or both. Forexample, a polynucleotide or a protein/peptide naturally present in aliving organism is not “isolated”, the same polynucleotide separatedfrom the coexisting materials of its natural state, obtained by cloning,amplification and/or chemical synthesis is “isolated” as the term isemployed herein. Moreover, a polynucleotide or a protein/peptide that isintroduced into an organism by transformation, genetic manipulation orby any other recombinant method is “isolated” even f it is still presentin said organism.

Modulation: Refers to the process by which a given variable is regulatedto a certain proportion.

Nucleic acid: Any DNA, RNA sequence or molecule having one nucleotide ormore, including nucleotide sequences encoding a complete gene. The termis intended to encompass all nucleic acids whether occurring naturallyor non-naturally in a particular cell, tissue or organism. This includesDNA and fragments thereof, RNA and fragments thereof, cDNAs andfragments thereof, expressed sequence tags, artificial sequencesincluding randomized artificial sequences.

Plant or Plant entity: refers to a whole plant or a part of a plantcomprising, for example, a cell of a plant, a tissue of a plant, anexplant, or seeds of a plant. This term further contemplates a plant inthe form of a suspension culture or a tissue culture including, but notlimited to, a culture of calli, protoplasts, embryos, organs,organelles, etc.

Polypeptide: means any chain of more than two amino acids, regardless ofpost-translational modification such as glycosylation orphosphorylation.

Promoter region: refers to a nucleotide sequence involved in theregulation of a specific gene. It is usually located at a 5′ position ofthe transcriptional start site. Typically, the promoter region includesthe TATA box and all regulatory elements (e.g. a silencing element whichnegatively regulates expression of the gene for proper regulation of aspecific gene.

Resistant or Tolerant. By resistant is meant a cell or organism (such asa plant) which exhibits substantially no phenotypic changes as aconsequence of an aggression by a pathogen (e.g. virus, fungus, insect,etc). By “tolerant” is meant a cell or organism which, although it mayexhibit some phenotypic changes as a consequence of aggression by apathogen, does not have a substantially decreased reproductive capacityor substantially altered metabolism.

SEBF nucleic acid: means any nucleic acid (see above) encoding a plantpolypeptide that is capable, among other things, of mediating repressionof a silencing element involves in plant defense mechanisms and capableof binding specifically onto the sequence BTGTCNC or YTGTCNC, the plantpolypeptide having at least 90%, preferably at least 95% and mostpreferably 100% identity or similarity to the amino acid sequence shownin SEQ. ID. NO:23. When referring to a plant SEBF nucleic acid, thenucleic acid set forth in SEQ. ID. NO: 21 encoding SEQ. ID. NO: 22 ismore particularly concerned. SEBF protein or SEBF polypeptide: means apolypeptide, a fragment thereof, or a functional SEBF homologue encodedby a SEBF nucleic acid as described above.

Similarity/Complementarity: In the context of nucleic acid sequences,these terms mean a hybridizable similarity under low, alternatively andpreferably medium and alternatively and most preferably high stringencyconditions, as defined below.

Specifically binds: means an antibody that recognizes and binds aprotein but that does not substantially recognize and bind othermolecules in a sample, e.g., a biological sample, that naturallyincludes proteins.

Substantially identical: means a polypeptide or nucleic acid exhibitingat least 50, 55, 60, 65, 70, 75%, preferably 80 or 85%, more preferably90, 95%, and most preferably 97 or 99% identity or similarity to areference amino acid or nucleic acid sequence. For polypeptides, thelength of comparison sequences will generally be at least 20 aminoacids, preferably at least 25, 30 or 40 amino acids, more preferably atleast 50 or 75 amino acids, and most preferably at least 100 aminoacids. For nucleic acids, the length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 100 nucleotides. Sequence identity is typically measuredusing sequence analysis software with the default parameters specifiedtherein (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Owl 53705). This software program matchessimilar sequences by assigning degrees of similarity to varioussubstitutions, deletions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

Stringency: For the purpose of defining the level of stringency,reference can conveniently be made to Maniatis et al. (1982) at pages387–389, and especially paragraph 11. A low stringency is defined hereinas being in 4–6×SSC/1% (w/v) SDS at 37–45° C. for 2–3 hours. Dependingon the source and concentration of nucleic acid involved in thehybridization, alternative conditions of stringency may be employed suchas medium stringent conditions which are considered herein to be1–4×SSC/0.5–1% (w/v) SDS at greater than or equal to 45° C. for 2–3hours or high stringent conditions considered herein to be0.1–1×SSC/0.1–1.0% SDS at greater than or equal to 60° C. for 1–3 hours.

Transformed or Transfected or Transgenic cell: Refers to introduction ofan exogenous nucleic acid, typically a gene or gene regulatory sequence,into a whole plant or a part thereof. By “transformation” is meant anymethod for introducing foreign molecules into a cell. Agrobacteriumtransformation, PEG treatment, lipofection, calcium phosphateprecipitation, electroporation, and ballistic transformation are just afew of the teachings which may be used.

Transgenic plant: any plant having a cell which includes a DNA sequencewhich has been inserted by artifice into the cell and becomes part ofthe genome of the plants which develops from that cell. Preferredtransgenic plants are those transformed with an exogenous nucleic acidintroduced into the genome of an individual plant cell using geneticengineering methods.

Vector: A self-replicating RNA or DNA molecule which can be used totransfer an RNA or DNA segment from one organism to another. Vectors areparticularly useful for manipulating genetic constructs and differentvectors may have properties particularly appropriate to expressprotein(s) in a recipient during cloning procedures and may comprisedifferent selectable markers. Bacterial plasmids are commonly usedvectors. Preferably, the vectors of the invention are capable offacilitating transfer of a nucleic acid into a plant cell and/orfacilitating integration into a plant genome.

Vegetal Host: refers to a cell, tissue, organ or organism comprisingchloroplasts and capable to perform photosynthesis. This term isintended to also include hosts which have been modified in order toaccomplish these functions. Algae and plants are examples of a vegetalhost.

Wound and stress inducible gene: a plant gene that is induced bywounding or by an abiotic stress such as ultraviolet light, drought,salinity.

B) General Overview of the Invention

The present inventors have now discovered that only nine nucleotides,i.e. the sequence GACTGTCAC (SEQ ID NO:26), are required for fullactivity of the silencing element (SE) of PR-10a gene.

The present inventors have also identified a nuclear factor (identifiedherein after as “SEBF”), that shows sequence specific binding to thesequence BTGTCNC (SEQ ID NO:23). Database searches revealed the presenceof this binding sequence in the promoter of numerous PR genes.

The present inventors have also found that the sequence BTGTCNC showshigh sequence similarity to the sequence of the auxin response element,strongly suggesting a role for this sequence and SEBF in the regulationof genes induced by the plant hormone auxin and its functional analogs.Furthermore, the inventors have demonstrated that SEBF can recognize andbind the sequence corresponding to the AuxRE of the GH3 gene of soybean.

The present inventors have also found that SEBF could be involved in theregulation of ethylene synthesis through the action of auxins since theSEBF binding element is present in the promoter of the ACC synthasegene, which is induced by auxins and encodes a key enzyme in thebiosynthesis of ethylene, another plant hormone.

The present inventors have also proceed to a molecular characterizationof SEBF. The SEBF protein was purified to homogeneity, partly chemicallysequenced and a cDNA encoding SEBF was isolated and sequenced. Thededuced amino acid sequence of SEBF shows a high degree of sequencesimilarity with chloroplast RNA binding proteins.

Subcellular partitioning of leaf cells demonstrates that SEBF is locatedin both chloroplasts and nuclei, suggesting functions in both cellularcompartments.

Overexpression of recombinant SEBF in a transient expression systemresults in SE-dependent transcriptional repression of a PR-10a-uidAreporter fusion, confirming the role of SEBF as a transcriptionalrepressor.

Using a two-hybrid system in the yeast, the present inventors furtheridentified proteins interacting with SEBF. Among those is Pti4, atranscription regulator of PR genes during the defense response inducedby plant pathogens. Interestingly, Pti4 is known to interact with theenzyme nitrilase, an enzyme involved in the biosynthesis of auxins.Furthermore, Pti4 is a member of the ethylene response element bindingproteins (EREBP) which are known to be involved in the plant responsesto ethylene. In addition, SEBF was found to interact with the ERF domainof Pti4 in two hybrid studies. The ERF domain is of particular interestbecause it is conserved among all ethylene response factors.

The present inventors have also demonstrated that an SE element presentin the promoter of the tobacco chitinase defense gene CHN50 is able tobind SEBF and that a mutation in this element that prevents binding ofSEBF derepresses (unblock) transcription. This result is of particularinterest because this promoter contains a GCC box, which is the bindingsite of Pti4, and is regulated by ethylene possibly through the bindingof ethylene response factors such as Pti4.

C) Nucleic Acids Comprising Specific Binding Sequences

According to a first aspect of the invention, there is provided anisolated or purified nucleic acid comprising a binding sequence ontowhich proteinic nuclear factor(s) specifically binds. According to apreferred embodiment, the binding sequence according to the invention isBTGTCNC (SEQ ID NO:23), B corresponding to any nucleotide other than A(i.e. T, C or G), and N corresponding to A, T, C or G. More preferably,the binding sequence according to the invention is YTGTCNC (SEQ IDNO:24), Y corresponding to a pyrimidine (i.e. T or C). The exact bindingsequence may vary depending on specific genes and organisms asexemplified herein after in Table 5.

According to a related aspect, there is provided there is provided anisolated or purified nucleic acid comprising a sequence that consists ofa gene regulatory element comprising a nucleic acid sequence that isessential for the full activity of the silencing element (SE) of PR-10a.According to one preferred embodiment, the gene regulatory elementconsists of a silencing element and it comprises sequence BTGTCNC (SEQID NO:23), and more preferably sequence YTGTCNC (SEQ ID NO:24).According to another preferred embodiment, the plant is potato and theessential sequence silencing element (SE) of PR-10a is CTGTCAC (SEQ IDNO:25 or 47).

The invention also concerns a DNA construct comprising the generegulatory element and/or the binding sequence described above, andgenetically modified plants entities comprising the gene regulatoryelement, the binding sequence and/or the DNA construct. In a preferredembodiment, the regulatory element is operatively linked to an induciblepromoter.

As it will be shown in the example hereinafter, the sequence BTGTCNC wasfound to be the specific binding sequence of a transcriptional repressorof the silencing element (SE) of PR-10a named SEBF. The sequence BTGTCNCit thus an important sequence involved in the modulation of the activityof genes incorporating this sequence into their promoter region.

Accordingly, the invention is also concerned with genetically modifiedvegetal hosts in which transcriptional activity of a gene associatedwith presence or absence of sequence BTGTCNC in a promoter region ofthis gene has been altered.

Therefore, a related aspect of the present invention concerns a methodfor altering gene expression in a plant. The method comprises the stepof altering in the plant binding of a nuclear DNA-binding protein tosequence BTGTCNC. Although the nuclear DNA-binding protein preferablyconsists of SEBF. A person skilled in the art will however understandthat SEBF is not the only proteinic nuclear factor that could bind tothe sequence BTGTCNC since other proteinic nuclear factor(s) could alsodo so. A non-limitative list of preferred nuclear DNA-binding proteinsincludes proteins having at least 48% identity or similarity to SEBF andmore particularly those given herein after in Table 6. The presentinvention also encompass sequences hybridizing under low, preferablymedium and more preferably high stringency conditions to the nucleicacid sequence BTGTCNC or to its complementary sequence.

D) SEBF and Other Polypeptides Binding to the Sequence BTGTCNC

According to further aspect the invention, there is provided an isolatedor purified nucleic acid molecule encoding a polypeptide that is capableof specifically binding the sequence BTGTCNC (SEQ ID NO:23), and morepreferably the sequence YTGTCNC (SEQ ID NO:24). There is also providedan isolated or purified polypeptide that is capable of specificallybinding the sequence BTGTCNC (SEQ ID NO:23), and more preferably thesequence YTGTCNC (SEQ ID NO:24).

The identity of the polypeptide as well as the binding sequence (seeTable 5) may vary depending on organisms and on specific genes for whichactivity is to be modulated. According to a preferred embodiment, thepolypeptide of the invention has an amino acid sequence at least 80 or85%, more preferably at least 90 or 95% and even more preferably 97, 99or 100% identical to the amino acid sequence shown in FIG. 5 (SEQ IDNO:22) and to functional homologues thereof. According to anotherembodiment, the polypeptide has an amino acid sequence encoded by anucleic acid at least 80 or 85%, more preferably at least 90 or 95% andeven more preferably 97, 99 or 100% identical to the nucleic acidsequence shown in FIG. 10 (SEQ ID NO:21), to its open reading frame(nucleotides 68 to 937), or to a fragment thereof. More preferably, thepolypeptide of the invention is also capable of modulating the activityof genes incorporating the sequence BTGTCNC or YTGTCNC into theirpromoter region. A person skilled in the art should be capable, withoutundue experimentation of determining the exact nucleotide sequencescoding for the polypeptide of the present invention.

According to a most preferred embodiment, the polypeptide of theinvention is a transcriptional repressor called “SEBF”. The sequence ofthe SEBF cDNA and predicted amino acid sequence is shown in FIG. 10 andin the “Sequence Listing” section. SEQ ID NO:21 corresponds to thepotato SEBF cDNA and SEQ ID NO:22 corresponds to the predicted aminoacid sequence of the protein.

The potato SEBF gene encodes a protein of 289 amino acids long. Insilico analysis indicates that potato SEBF protein has the followingfeatures: it has a molecular weight of about 30 810 g/mol, anisoelectric point of about 4.6; an instability index of about 49; analiphatic index of about 66.8; and a grand average of hydropathicity(GRAVY) of about −0.5. It further comprises many potentialphosphorylation sites (18 Ser, 3 Thr, and 5 Tyr); and also 10 potentialN-glycosylation sites. On an SDS PAGE under denaturing conditions, ithas an apparent molecular weight, of about 28 to about 29 kDa. It alsohas the biological activity of a transcriptional repressor.

Blast searches were made to identify sequence identity SEBF and otherexisting sequences. Table 1 herein after provides a list of sequenceshowing similarity to SEBF (nucleotide vs nucleotide). Table 2 providesa list of proteins homologous to SEBF (protein vs protein) and Table 3provides a list of predicted proteins showing similarities to SEBF(protein vs translated nucleotide).

The polypeptides and nucleic acid molecules of the present invention,and more particularly SEBF and/or is binding sequence, may be preparedby any suitable process. They may for instance be obtained by chemicalsynthesis when appropriate. They may also be prepared using biologicalprocesses involving cloning or expression vectors. Such vectors wouldcomprise a polynucleotide sequence incorporating the nucleic acidmolecule of interest such as and/or comprise a polynucleotide sequenceencoding for the peptide of interest. Therefore, the present inventionencompass such cloning or expression vectors and more particularly thoseencoding SEQ ID NO:22 and those comprising nucleotides 68 to 937 of SEQID NO:21. In addition, standard techniques, such as the polymerase chainreaction (PCR) and DNA hybridization, may be used to clone additionalSEBF homologues in other plant species.

Therefore, in a related aspect, the invention is directed to a methodfor producing, in vitro, a polypeptide capable of specifically bindingthe sequence BTGTCNC (SEQ ID NO:23), and more preferably the sequenceYTGTCNC (SEQ ID NO:24), and having preferably the biological activity ofa transcriptional repressor. This method comprises the step of: 1)culturing in vitro, in a suitable culture medium, a cell incorporatingan expression vector as described previously; and optionally 2)collecting in the culture medium polypeptides produced by these cells.Methods for producing such genetically modified cells and methods forusing these cells in the production of proteins/peptides are well knownin the art and will no be described in detail herein.

TABLE 1 Nucleotide sequence showing similarity to SEBF (nucleotide vsnucleotide) NCBI acc. Identity Sequence number raw percent Expect¹ mRNAfor cpRBP30, GI: 19707 777/885 87 0.00E+00 Tobacco mRNA for cp29A, GI:19753 481/544 88 1.00E−54 Tobacco mRNA for cp31, GI: 19709 434/456 952.00E−58 Tobacco mRNA for cp29B, GI: 14134 345/380 91 1.00E−44 TobaccoAt2g37220 gene, GI: 13877808 130/154 84 1.00E−22 Arabidopsis mRNA forcp29 GI: 18409841 126/155 81 3.00E−11 (At3g53460), Arabidopsis mRNA for24 kDa RBP, GI: 1015369  93/107 87 9.00E−05 Spinach ¹The expect valuerepresents the possibility of choosing a particular sequence randomly

TABLE 2 Proteins similar to SEBF Identity Similarity Protein NCBI acc.number raw percent raw % Expect¹ cp29A, Tobacco GI: 12230584 191/230 83198/230 86 1.00E−118 30 kDa ribonucleoprotein, Tobacco GI: 1350820186/232 80 193/232 83 4.00E−88 cp29B, Tobacco GI: 12230585 173/245 70188/245 76 3.00E−83 31 kDa ribonucleoprotein, Tobacco GI: 1350821172/245 70 189/245 76 2.00E−73 Putative RNA binding protein, ArabidopsisGI: 15228102 148/219 67 173/219 78 2.00E−73 Putative RNA bindingprotein, Rice GI: 18921322 137/221 61 166/221 74 8.00E−68 RNA bindingprotein, Spinach GI: 7446360 142/224 63 162/224 71 1.00E−60 cp29,Arabidopsis GI: 681904 144/265 54 167/265 62 2.00E−59 RNA bindingprotein, Avocado GI: 19032260 113/225 50 147/225 65 2.00E−48 cp31,Arabidopsis GI: 1076305 112/226 49 146/226 64 3.00E−48 RNP-D,Arabidopsis GI: 629557 112/222 50 144/222 64 5.00E−47 RNP-T, ArabidopsisGI: 15233980 112/222 50 144/222 64 1.00E−46 28 kDa ribonucleoprotein,Tobacco GI: 133246 102/219 46 142/219 64 1.00E−46 RNA binding protein,Pea GI: 7446357 110/213 51 138/213 64 2.00E−46 RNA binding protein 2,Arabidopsis GI: 475719 112/222 50 144/222 64 2.00E−46 cp31, Barley GI:7446358 114/220 51 145/220 65 6.00E−46 Ps16, Wheat GI: 7446356 113/22151 146/221 65 5.00E−45 DNA binding protein, Arabidopsis GI: 99684112/222 50 144/222 64 9.00E−45 RNA binding protein, Ice plant GI:1076251 112/228 49 140/228 61 2.00E−44 RNA protein-like, Arabidopsis GI:15240641 110/225 48 141/225 61 5.00E−44 28 kDa ribonucleoprotein,Spinach GI: 133247 116/217 53 140/217 64 1.00E−43 nucleic acid-bindingprotein, Maize GI: 100903 112/221 50 143/221 64 7.00E−42 CEBP-1, Clovepink GI: 7446355 107/245 43 140/245 56 5.00E−41 RNA binding protein 3,Arabidopsis GI: 475720  93/172 54 117/172 67 3.00E−40 RNP1, Kidney beanGI: 1076509  89/222 40 128/222 57 1.00E−34 cp33, Arabidopsis GI: 681912 87/222 39 138/222 61 1.00E−34 ribosomal protein CEP52, Arabidopsis GI:17064758  87/222 39 137/222 61 3.00E−34 cp33, Tobacco GI: 133249  90/22839 140/228 60 3.00E−34 cp33, Barley GI: 7446339  89/226 39 130/226 571.00E−33 RNA binding protein, Fava bean GI: 7446361  87/226 38 132/22657 2.00E−32 Ribosomal protein 2, Spinach GI: 7578881  76/203 37 118/20357 3.00E−27 NSR1, Yeast GI: 253181  60/188 31 103/188 53 6.00E−21 ¹Theexpect value represents the possibility of choosing a particularsequence randomly

TABLE 3 Predicted proteins showing similarity to SEBF (protein vstranslated nucleotide) Identity Similarity Protein NCBI acc. number rawpercent raw percent Expect¹ mRNA for cpRBP30, Tobacco GI: 19707 210/29371 220/293 74 1.00E−102 mRNA for cp29A, Tobacco GI: 19753 208/292 71216/292 73 1.00E−101 mRNA for cp31, Tobacco GI: 19709 173/254 68 191/25475 3.00E−181 At2g37220, Arabidopsis GI: 16323481 152/248 61 181/248 722.00E−76 mRNA for cp29 (At353460), Arabidopsis GI: 681903 151/282 53173/282 60 6.00E−68 mRNA for PCO130298, Maize GI: 21206992 134/203 66159/203 78 1.00E−67 mRNA for PCO127552, Maize GI: 21208658 129/192 67153/192 79 3.00E−65 mRNA for 24 kDa RBP, Spinach GI: 1015369 133/193 68148/193 75 5.00E−63 mRNA for cp31AHv, Barley GI: 3550466 106/191 55135/191 70 1.00E−48 mRNA for cp31, Arabidopsis GI: 681907 104/192 54132/192 68 5.00E−48 mRNA for RNP-T, Arabidopsis GI: 18416422 104/192 54132/192 68 5.00E−48 mRNA for Ps16, Wheat GI: 2443389 105/191 54 134/19169 5.00E−48 mRNA for rbp33, Avocado GI: 19032259 103/192 53 132/192 682.00E−47 mRNA for ribonucleoprotein, Pea GI: 2330646 106/195 54 130/19566 6.00E−47 mRNA for CL2457_1, Maize GI: 21216451 103/189 54 132/189 694.00E−46 mRNA for cp28, Tobacco GI: 19749  96/199 48 131/199 65 2.00E−45¹The expect value represents the possibility of choosing a particularsequence randomlyE) SEBF Antibodies

The polypeptides and polynucleotides of the invention may also be usedfor producing polyclonal or monoclonal antibodies capable of recognizingand binding the same. Accordingly, the invention also features apurified antibody (monoclonal or polyclonal) that specifically binds toa SEBF protein and/or a functional homologue thereof, such as SEBFhomologous proteins in other plants species.

The Exemplification section of the application describes the preparationof a SEBF antibody. This antibody was also tested against a tobacco leafextract and two strong bands of 28 and 29 kDa (probably corresponding tocp29A and cp29B respectively) were revealed. A preliminary test alsosuggests that the antibody the prepared SEBF antibody also recogniseshomologous proteins in Arabidopsis thaliana.

The antibodies of the invention may be prepared by a variety of methodsusing the SEBF proteins or polypeptides described above. For example,the SEBF polypeptide, or fragments thereof, may be administered to ananimal in order to induce the production of polyclonal antibodies.Alternatively, antibodies used as described herein may be monoclonalantibodies, which are prepared using hybridoma technology (see, e.g.,Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., 1981).

SEBF antibodies may be used for preventing the action of SEBF in theplant. For example, a transgenic plant could be made which expresses anSEBF antibody (ex a single-chain antibody). Binding of this antibody toSEBF would inhibit its activity, therefore leading to derepression ofgenes under the control of SEBF. Accordingly, preferred antibodies are“neutralizing” antibodies. By “neutralizing” antibodies is meantantibodies that interfere with any of the biological activities of theSEBF polypeptide, particularly the ability of SEBF to repress geneactivity. The neutralizing antibody may reduce the ability of SEBFpolypeptides by preferably 50, 55, 60 or 65%, more preferably by 70, 75,80 or 85%, and most preferably by 90, 95, 99% or more. Any standardassay, including those described herein, may be used to assesspotentially neutralizing antibodies. Once produced, monoclonal andpolyclonal antibodies are preferably tested for specific SEBFrecognition by Western blot, immunoprecipitation analysis or any othersuitable method.

Alternatively, the antibody could be specifically targeted to theplastids by translational fusion with a plastid transit peptide,therefore leading specifically to inhibition of plastidic functions ofSEBF. Time of expression of the antibody could be controlled by placingits expression under the control of an inducible promoter, such as apromoter inducible by treatment with estrogens. The present inventiontherefore encompass such antibodies and methods for using the same.Methods for producing antibodies are well known in the art.

F) Genetically Modified Cells and Plants

The invention is also concerned with vegetal hosts, particularly plants,genetically modified for reducing (or increasing) the regular biologicalactivity associated with the presence (or absence) into theregulatory/promoter region of the genes of the plant, of the sequenceBTGTCNC (SEQ ID NO:23), and more preferably the sequence YTGTCNC (SEQ IDNO:24). Depending on specific uses as it will be described hereinafter,it may be advantageous to have plants in which at least some cells havebeen genetically modified so that the sequence BTGTCNC is inactive(mutation, deletion, etc). In other cases, it may be preferable toinsert the sequence BTGTCNC or YTGTCNC into specific genes of theplants.

The invention is also concerned with cells and organisms geneticallymodified for expressing higher (or lower) levels of polypeptide(s) thatis capable of specifically binding the sequence BTGTCNC (SEQ ID NO:23),and more preferably the sequence YTGTCNC (SEQ ID NO:24) (i.e. modulationof the cells or organisms endogenous levels). Depending of specific usesas it will be described hereinafter, it may be advantageous to havecells, and more particularly plant cells, that have been geneticallymodified to increase the levels of at least one polypeptide capable ofspecifically binding the sequence BTGTCNC or YTGTCNC, such as the SEBFprotein or of a functional SEBF homologue. In other cases, it may bepreferable to reduce the levels of such polypeptide(s).

Specific examples on how to prepared and/or used such geneticallymodified plants are described in Section H.

G) Compositions

The invention also relates to compositions for 1) modulating in plantsthe regular biological activity associated with the presence (orabsence) of the sequence BTGTCNC (SEQ ID NO:23), and more preferably thesequence YTGTCNC (SEQ ID NO:24) and/or 2) modulating in plants thelevels of expression of the polypeptide(s) capable of specificallybinding the sequence BTGTCNC (SEQ ID NO:23), and more preferably thesequence YTGTCNC (SEQ ID NO:24) (i.e. modulation of the plant endogenouslevels).

Such a composition would comprise an effective amount of at least onecompound that is capable of, directly or indirectly, achieving one orboth of the above mentioned desired effect, in combination with adiluent or a carrier. Herbicides are example of small molecules thataffect plant physiology and development by binding and inhibiting thefunction of specific plant proteins. The compound(s) and its amountwould be selected such that, following application of the composition ofthe invention, the desired modulation occurs into at least some of thecells of the plant when compared to a corresponding plant in the absenceof the composition. More specific but non restrictive examples ofsuitable compositions according to the invention will be givenhereinafter in Section H.

Plants treated with suitable compositions could show induced resistanceto pathogens. Depending on time of treatment, the compositions couldalso be used to increase fruit maturation and number, or to inducesenescence.

The carrier or diluent can be a solvent such as water, oil or alcohol.The composition may also comprise others active agents such asfertilizers and growth regulators. The inducing composition may also beformulated with emulsifying agents in the presence or absence offungicides or insecticides, if required. The precise amount of compoundemployed in the practice of the present invention will depend upon thetype of response desired, the formulation used and the type of planttreated.

H) Specific Examples of the Uses of the Invention

The following examples are illustrative of the wide range ofapplicability of the present invention and are not intended to limit itsscope.

I-Increase Plant Resistance or Tolerance to Pathogens

Since SEBF is a transcriptional repressor of the defense response, andthat its binding site has been found in numerous defense genes whoseexpression has been shown to lead or correlate to the defense responsein plants, inhibition or reduction of SEBF accumulation (and/or of afunctional SEBF homologue) and/or inhibition or reduction of the regularprotein biological activity could increase the plant resistance topathogens or to a faster induction of its defense mechanisms.

Such reduction of SEBF endogenous levels and/or reduction of SEBFbiological activity (and/or levels or activity of a functional SEBFhomologue) could be achieved in all plant species, by a number of means,including: expression in a transgenic plant of an antisense copy of theSEBF gene, or part of the gene; by expression of a sense copy thatinduces a co-suppression mechanism of SEBF; by production of a knock outof the SEBF gene by inserting therein a foreign DNA molecule; bychemical mutagenesis of the SEBF gene; by expression of a ribozymecleaving SEBF mRNA; or by any other mean leading to the reduction ofSEBF gene expression, accumulation of SEBF mRNA, and other suitablemethods.

Similarly, SEBF binding site could also be genetically modified so thatthe sequence BTGTCNC (or YTGTCNC) becomes inactive (removal, mutation,deletion, etc).

Addition of the sequence BTGTCNC (or YTGTCNC) into the promoter of agene of interest would reduce the level of expression of this specificgene. In some cases, for instance where the endogenous SEBF activitywould be too weak, overexpression of SEBF could be required, in additionto the insertion of the SE sequence, for proper gene control under SE.On the other hand, removal of the sequence BTGTCNC (or YTGTCNC) wouldgive higher sequence of expression. Such promoter alterations would beuseful in plants where the level of SEBF (and/or functional SEBFhomologue) cannot be altered. For example, removal of this sequence froma defense gene and the introduction of this modified gene in transgenicplants would lead to constitutive expression of this gene and to higherresistance to pathogens.

Overexpression of SEBF (and/or of a functional SEBF homologue) couldlead to plants with reduced resistance to infection. This property couldalso be useful to control transgenic plants dissemination. For example,a transgenic plant engineered to express medicinal compounds could befurther genetically modified so that it becomes hypersensitive topathogens. This would allow easy elimination of these plants incultivated fields by treatment of these plants with a pathogen known notto infect wild type plants.

II. Modifying Plant Responses to Auxins and Ethylene

i) Auxins

Auxins are diffusible growth-promoting plant hormones. The primary auxinpresent in most plant is indole-3-acetic acid (IAA). It is active in lowconcentrations and synthetic analogs, 2,4-dichlorophenoxyacetic acid(2,4-D) and naphtalene-1-acetic acid (NAA), are used in agriculture toinduce rooting and to promote the set and development of fruit. At highconcentrations auxins inhibit plant growth and are often used asherbicides. Indole-3-acetic acid is synthesized mainly from L-tryptophanbut an alternative route involves the conversion ofindole-3-acetonitrile through the action of nitrilases. Highconcentrations of auxins are generally accompanied by an increased rateof ethylene biosynthesis due to the activation by auxins of the ACCsynthase gene, which controls the production of ethylene.

As it will be shown in Example 1 hereinafter, the auxin response elementoverlaps with that of SEBF. This suggest that SEBF (and/or functionalSEBF homologues) is able to bind to the auxin response element.Therefore SEBF and its functional homologues may be involved in therepression of genes controlled by auxin. Therefore, induction of genescontrolled by auxins could be increased or decreased by modulating theendogenous levels of SEBF (and/or levels of functional SEBF homologues).SEBF levels could be decreased by one of the strategy described inSection I hereinabove. Plants with lower levels of SEBF or with a lowerSEBF activity could have an increased sensitivity to endogenous auxinsand exhibit an increased growth, rooting and fruit number as compared toother plants, and vice versa.

Plants with lower endogenous levels of SEBF (and/or functional SEBFhomologues) or with a lower SEBF activity could also become moresensitive to exogenous treatment with auxin and its analogs. Thereforeless of auxin-based herbicides could be used to eliminate these plantsas compared to other plants. This property could also be useful tocontrol transgenic plants dissemination. For example, a transgenic plantengineered to express medicinal compounds could be further geneticallymodified so that it becomes hypersensitive to auxinic herbicides. Thiswould allow easy elimination of these plants in cultivated fields byspraying with low doses of the auxinic herbicide. Alternatively, plantswith higher endogenous levels of SEBF such as transgenic plantsover-expressing the SEBF gene could be less sensitive to auxin andanalogs thereof, thereby conferring auxinic herbicide resistance tothese plants.

Of course, insertion, deletion, mutation, of SEBF binding site sequencecould also be considered to increase/decrease SEBF biological activityand modulate accordingly plants auxinic response.

ii) Ethylene

Ethylene influences seed germination, root and shoot growth, flowerdevelopment, senescence and abscission of flowers and leaves, and fruitripening. Ethylene is what causes fruits to over ripen and this is themajor cause of fruit decay after harvest. ACC synthase, which is inducedby auxins, catalyses the rate limiting step in ethylene biosynthesis.

As it will be shown in Example 1 hereinafter, the SEBF binding site ispresent in the promoter of the ACC synthase gene in potato and innumerous plant species (see Table 5). This strongly suggests that theauxin induction of the ACC synthase gene is regulated by SEBF. Thishypothesis is also supported, as it will be shown in the examplehereinafter, by the interaction of SEBF with Pti4, a member of theethylene response element binding proteins family.

Plants with higher endogenous levels of SEBF (and/or of a functionalSEBF homologue), such as transgenic plants over-expressing SEBF, shouldtherefore be capable of down regulating ACC synthase and be capable ofreducing the production of ethylene. Reduction of ethylene productionwould in turn delay fruit ripening and protect fruits againstover-ripening.

On the other hand, plants with lower endogenous levels of SEBF or lowerSEBF activity such as plants expressing an antisense copy of the SEBFgene, or part of it, could exhibit an early fruit maturation.

Addition of the sequence BTGTCNC (or YTGTCNC) into the promoter of agene of interest would reduce the level of expression of this specificgene. On the other hand, removal of the sequence BTGTCNC (or YTGTCNC)would give higher sequence of expression. Such promoter alterationswould be useful in plants where the level of SEBF cannot be altered. Forexample, removal of the BTGTCNC (or YTGTCNC) sequence from the promoterof the ACC synthase gene could lead to increase production of ethylene.After modification, the modified promoter and the coding sequence wouldbe introduced in plants to create transgenic plants.

III. Chloroplastic Functions

Lowering the concentration of SEBF in the chloroplast or reducing SEBFbiological activity in this organelle could result in marked phenotypicmodifications of the plant. As it will be shown in Example 1hereinafter, SEBF homologues have been shown to interact and stabilizeRNA in plastids. Accordingly, it should be possible to modulate theexpression of the plastid mRNAs onto which SEBF binds by modulating SEBFlevels (and/or levels of functional SEBF homologues). This could giveplants with a better photosynthetic or carbon fixation rate or plantsbetter adapted to their environment.

IV. Protein Modules and Other Research Tools

As it will be shown in the example hereinafter, SEBF can be localized intwo different compartments: the nucleus and the plastid. This indicatesthat SEBF amino acid sequence comprises a first “tag” (amino acids 1 to59 of SEQ ID NO:22) which contains the localization information todirect SEBF to the plastid and a second “tag” to direct SEBF to thenucleus. Such tags could be used to direct other proteins to the plastidor nucleus by making a translational fusion between this tag(s) and theprotein to be targeted into these cellular compartments.

Preliminary evidence also suggests that SEBF is released from its DNAbinding element upon wounding or pathogen challenge of the plant. Thisfeature could be used to create promoter systems tightly regulated inabsence of inducing events. This could be achieved by inserting the SEBFbinding sequence inside known inducible promoters. Such geneticallymodified promoters would then require two inducible events (one beingthe newly introduce SEBF binding sequence) in order to activateexpression of a related gene.

V. Screening for Molecules Modulating SEBF Binding

Since SEBF is a binding molecule, this characteristic could be used forscreening and/or identifying novel compounds that: inhibit the bindingof SEBF to its DNA element (the sequence BTGTCNC); that inhibit thebinding of SEBF to plastid or nuclear mRNAs; that inhibit theinteraction of SEBF with another protein; and/or that negatively affectSEBF tridimensional structure (e.g. post-translational modifications).

Inhibition of SEBF binding activity could be measured in vitro byelectromobility shift assays, by in situ by treating a plant withvarious chemicals, or by any other suitable method known in the art.

Compounds capable of inhibiting SEBF binding activity would have manyuses as describe hereinbefore, and more particularly for increasing aplant resistance to pathogen or induce its defense response mechanism;for increasing a plant sensitivity to endogenous auxins and therebyincrease growth, rooting and fruit number of the plant; increasing aplant sensitivity to auxin-based herbicides; and induce fruitmaturation.

Similarly, compounds capable of increasing SEBF binding activity orwould have many uses as described hereinbefore, and more particularlyfor reducing a plant sensitivity to auxin and analogs thereof (therebyconferring auxinic herbicide resistance to these plants); for delayingfruit ripening and protecting fruits against over-ripening caused byproduction of ethylene.

EXAMPLE 1

The following example is illustrative of the wide range of applicabilityof the present invention and is not intended to limit its scope.Modifications and variations can be made therein without departing fromthe spirit and scope of the invention. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferred methodsand materials are described.

a) Introduction

A variety of defense specific events are induced in plants in responseto pathogen infection, including the production of reactive oxygenspecies, activation of G-proteins, reinforcement of the cell wall andthe induction of signal transduction cascades leading to thetranscriptional activation of defense genes (Dixon et al., 1994;Blumwald et al. 1998). Although key components of the signaling cascadesare being discovered, few transcription factors have been identifiedthat integrate these signals at the transcriptional level.

The well characterized PR genes induced by pathogen invasion provideexcellent models to study transcriptional regulation of defense genes.PR genes are subdivided into 11 classes based on sequence similarity(Van Loon et al., 1994). The PR-10 gene family is subdivided into twogroups and encodes small, acidic intracellular proteins of 15–18 kD(Osmark et al, 1997). Although Rnase activity has been demonstrated forPR-10 proteins, they do not possess any sequence similarity withclassical Rnases (Moiseyev et al., 1994; Swoboda et al., 1996). Inpotato, three members of this family have been identified of whichPR-10a is the best characterized.

Expression studies have identified cis-acting elements involved inPR-10a gene regulation. An elicitor response element (ERE) locatedbetween nucleotides −135 and −105 is essential and sufficient forelicitor induced gene expression (Matton et al, 1993; Després et al.,1995; Desveaux et al, 2000). PBF-2, a single-stranded DNA bindingfactor, appears to play a role in activation of PR-10a from the ERE(Després et al., 1995; Desveaux et al, 2000). Although the presence ofthe ERE is sufficient for PR-10a activation, removal of the silencingelement (SE), located between −52 and −27, leads to further activation,suggesting that this element participates, with the ERE, in theregulation of PR-10a (Matton et al, 1993; Després et al., 1995).

In the present invention, we demonstrate, using transient expressionstudies, that only 9 nucleotides containing the sequence GACTGTCAC arerequired for full SE activity. We have also identified a nuclear factor(SEBF), that shows sequence specific binding to the sequence BTGTCNC.Database searches revealed the presence of this sequence in the promoterof numerous PR genes. This sequence also shows high sequence similarityto the sequence of the auxin response element, suggesting a role forthis sequence and SEBF in the regulation of genes induced by the planthormone auxin and its functional analogs. In particular, the SEBFelement is present in the promoter of the ACC synthase gene, which isinduced by auxins and encodes a key enzyme in the biosynthesis ofethylene, another plant hormone. Therefore SEBF could be involved in theregulation of ethylene synthesis through the action of auxins.

SEBF was purified to homogeneity from potato tubers and sequencing ofthe N-terminus of the protein led to the isolation of a cDNA encodingSEBF. The deduced amino acid sequence shows a high degree of sequencesimilarity with chloroplast RNA binding proteins. Subcellularpartitioning of leaf cells demonstrates that SEBF is located in bothchloroplasts and nuclei, suggesting functions in both cellularcompartments. Overexpression of recombinant SEBF in a transientexpression system results in SE-dependent transcriptional repression ofa PR-10a-uidA reporter fusion, confirming the role of SEBF as atranscriptional repressor. Using the yeast two-hybrid system, weidentified proteins interacting with SEBF. Among those is Pti4, atranscription regulator of PR genes during the defense response inducedby plant pathogens. Pti4 is known to interact with the enzyme nitrilase,an enzyme involved in the biosynthesis of auxins. Furthermore, Pti4 is amember of the ethylene response element binding proteins (EREBP) whichare known to be involved in the plant responses to ethylene.

b) Materials and Methods

All standard molecular biology procedures were done according toSambrook et al. (1989).

Plant Material

Potato tubers (Solanum tuberosum cv. Kennebec) were obtained from theQuebec Ministry of Agriculture “Les Buissons” Research Station(Pointe-aux-Outardes, Canada). Tubers were stored in the dark at 4° C.and brought to room temperature 24 hr before use. Leaf mesophyllprotoplasts were isolated from 4–5 weeks old potato plants grown ingrowth chambers as described by Magnien et al. (1980). Potato leafs wereisolated from plants grown in an environmental growth chamber (Conviron,Winnipeg, Canada) under long day photoperiod conditions.

GUS Reporter Constructs and Analysis

The wild type (WT) construct was created by PCR amplification of thePR-10a promoter fragment of plasmid p-135 (Matton et al., 1993) usingPCR oligol (GCCMGCTTTAGATAAAATGACACAAATGTCAAAAATGG; SEQ ID NO:27) andoligo2 (CCACCCGGGGATCCAGCTTTGMC; SEQ ID NO:28) to replace the XbaI siteat position −135 by a HinDIII site. After digestion with HinDIII andBamHI, the fragment was inserted into pBI201. Mutant m1 was describedpreviously (pLP9, Desveaux et al., 2000). Mutants m3, m4 and m5 werecreated by PCR using mutated oligonucleotides (FIG. 2A) and PCR oligo 2.The PCR fragments were cleaved with XbaI and BamHI and inserted into thewild type plasmid described above. For mutant m2, a HinDIII/NcoI PCRfragment was synthesized using oligo 1 and oligo 3 (CAGTCCATGGTTAAATCMC;SEQ ID NO:29). Then a NcoI/BamHI PCR fragment was synthesized usingoligo 2 and oligo 4 (TAACCATGGACTGTCACTTG; SEQ ID NO:30). Ligation ofthese fragments into pBI201 cleaved with HinDIII and BamHI createdmutant m2 (SEQ ID NO:2). The following primers were used to amplify theCHN50 promoter from tobacco: 5′-GCCGCMGTTTTCTGCAGTGTTTTTGCTC-3′ (SEQ IDNO:31) and 5′-GGTTTGGATCCAGTAGTAAAGTGGCGA-3′ (SEQ ID NO:32). Theamplified DNA was digested with PstI and BamHI and then inserted intothe same sites of plasmid pBI201. Mutations in the promoter were donewith the ExSitem PCR-based site-directed mutagenesis kit (Stratagene)using the oligonucleotides shown in FIG. 12C and oligonucleotide CHN50R5′-ATGTCTACTCCTGCGCTCATT-3′ (SEQ ID NO:33). Amplifications were carriedout in a Whatman/Biometra Tgradient™ thermal cycler. All constructs weresequenced to ensure that no mutations were inserted by PCR. Thetransient expression system was previously described (Mafton et al.,1993). All values were corrected for the efficiency of electroporationusing luciferase activity resulting from the coelectroporation of 1 μgof plasmid pWB216 (Barnes, 1990).

The coding sequence for the full length SEBF was inserted into pBI223Dwhich contains a double CaMV ³⁵S enhancer. For transrepression studies 5μg of expression vector was added to the assays. The control vectorexpressed a human fusion protein containing FK506-binding protein (FKBP;Pelletier et al., 1998) under the same promoter.

Preparation of Extracts

Crude nuclear extracts were prepared as follows: 200 g of potato tubersor 75 g of leaves were homogenized in a blender at maximum speed for 1min in 250 ml of cold NEBH (10 mM PIPES-KOH, pH 6.0, 1 M 2-methyl-2,4pentanediol, 0.15 mM spermine, 0.5 mM spermidine, 10 mM MgCl₂, 14.3 mMP-mercaptoethanol, 0.1 mM PMSF). After decantation at 4° C. for 5 min,the homogenate was filtered through 5 layers of Miracloth™ under vacuum.The filtrate was centrifuged at 5,000 RPM for 5 min in a Sorval GSArotor to pellet the nuclei. The supernatant served as the cytosolicfraction. Nuclei were washed three times in NP-40 buffer (10 mM MES-NaOHpH 6.0, 260 mM sucrose, 10 mM NaCl, 1 mM EDTA, 0.15 mM spermine, 0.5 mMspermidine, 14.3 mM P3-mercaptoethanol, 0.1% BSA and 1% NP40) to removeorganelle and cytoskeleton contamination. The final pellet wasresuspended in 5 ml of 200 mM NaCl SEBF binding buffer (SBB: 10 mMTris-HCl, pH 7.5, 1 mM EDTA, 14.3 mM β-mercaptoethanol, variable NaClconcentrations). Nuclei were ruptured by sonication and the lysatecentrifuged at 15,000 RPM for 45 minutes. The supernatant was eitherused immediately for SEBF purification or stored at −70° C. in 10%glycerol. In such conditions SEBF binding activity was stable formonths. Chloroplasts were prepared by the method of Gegenheimer (1990).

Determination of Enzymatic Activities in Extracts

Chlorophyll content was measured according to Arnon (1949). Alkalinepyrophosphatase, nitrite reductase and alcohol dehydrogenase weremeasured according to Gross and ap Rees (1986), Hucklesby et al. (1972)and Smith and ap Rees (1979), respectively.

Electrophoretic Mobility Shift Assays (EMSA)

Protein extracts (in 200 mM NaCl SBB) were mixed with 20,000 CPM of ³²Pend-labeled probe. The binding reaction (40 μl) contained 100 ng ofpoly(dI-dC) to eliminate non-specific binding. The reaction was left onice for 15 min. Electrophoretic separation of the complexes was achievedby loading the mixture on a 5.7% polyacrylamide gel prepared inTris-glycine buffer (25 mM Tris, 195 mM glycine) and applying 200 V for2.5 h at 4° C. The gel was exposed overnight to a Kodak X-omat AR filmat −70° C.

Purification of SEBF from Nuclear Extracts

Crude nuclear extracts were loaded onto a 3 ml Q-sepharose™ FPLC™(Amersham-Pharmacia Biotech) column equilibrated in 200 mM NaCl SBB. Thecolumn was washed with 10 volumes of 200 mM NaCl SBB followed by 15volumes of 300 mM NaCl SBB. SEBF was eluted in 4 ml of 400 mM NaCl SBB.The eluate was then submitted to two rounds of affinity purification.The affinity beads were prepared by coupling a biotinylated WT SEoligonucleotide to streptavidin coated paramagnetic beads (Sigma)according to Desveaux et al. (2000). The beads were washed twice with400 mM NaCl SBB prior to use. To 1 ml of eluate was added 20 μl of EDTA500 mM pH 8.0, 10 μg of poly(dI-dC) and 5 μl of NP-40. The mixture wasadded to the affinity beads and left on ice for 15 min with sporadicmixing. After separation of the beads from the solution using a magnet,the beads were washed in 1 ml of 200 mM NaCl SBB followed by a 1 ml washwith 1 M NaCl SBB. SEBF was eluted from the beads by two subsequent 0.1ml of 2 M NaCl SBB and incubated at 37° C. for 5 min. The eluatecontaining SEBF was desalted using Ultrafree 4™ centrifugal filter 10 KBioMax™ (Millipore, Bedford, USA). The equivalent of 1 kg of tubers waspooled and acetone precipitated. The pellet was resuspended in Laemmlisolubilizing buffer (Laemmli 1970) and loaded on a 12%SDS-polyacrylamide gel. The proteins were transferred to a PVDF membrane(BIO-RAD) and stained with Coomassie blue. Bands were cut and sent forN-terminal analysis at the Eastern Quebec Proteomics Core Facility(Ste-Foy, Canada). The sequenceVTLSDFDQIEEVEAGDDDEEEGGLSDEAGASYEERN?NPDL; SEQ ID NO:34) was obtainedfrom amino terminal analysis of the 29 kD band. The southwesternanalysis was done according to Vinson et al. (1988).

Cloning of SEBF

A degenerate oligonucleotide (GTKACVVYTITCIGATTTYGAYCA; SEQ ID NO:35)corresponding to the N-terminal sequence of SEBF and a cs-RBD specificprimer (ARRTTWCCIACRMRATYTT; SEQ ID NO:36; Mieszczak et al., 1992) (Icorresponding to an inosine) were used to generate a 141 bp genomic PCRfragment of SEBF. The specific primer GTTTGAGTGATGAAGGTGC (SEQ ID NO:37)was derived from the sequence of the 141 bp fragment and used to probe apotato cDNA bank (Matton and Brisson, 1989) using the method proposed byIsrael (1993). Twenty-three pools of 18,000 phages were used in PCRreactions using the gene specific primer and the M13 universal primer.Two pools gave a PCR fragment of approximately 1,000 bp. These poolswere further diluted and the procedure repeated until it was possible toisolate a single plaque. A positive clone was excised by using theExAssistm system (Stratagene), according to the manufacturersinstructions. The clone was sequenced on both strands withThermosequenasem (Amerham-Pharmacia Biothech) in a cycle-sequencingreaction that was then processed on a LI-COR™ automated sequencer(LI-COR, Lincoln, USA). SEBF nucleotide and amino acids sequences (SEQID NOs:21 and 22) were published by the present inventors on Dec. 10,2001 in GenBank™ under accession No. AF389431.

Southern Analysis and Genomic Analysis

Procedures for the extraction of genomic DNA and Southern blotting wereas described by Ausubel et al. (2001). The following primers weredesigned according to the predicted position of the introns in thetobacco and Arabidopsis SEBF homologues (Ye et al., 1991; Ohta et al.,1995) and used for PCR analysis of the genomic DNA:

T: GCCGTTCTGTCTTCACAATTCTTTTGCTTC; (SEQ ID NO:38) U:CAACATCTCCAGCACGCTCAAAAAGCTCAG; (SEQ ID NO:39) V:CCTCTGCTTCTTCCTGTAAGCTTGTCATAG; (SEQ ID NO:40) W:CAGTTGAAGCCGCCTGTCAACAATTTAATG; (SEQ ID NO:41) X:TTGACGGGAGGGCACTGAGGGTGAATTCTG; (SEQ ID NO:42) Y:GCTTTCAATTGCAT-CGTTGACCTCCTTAG; (SEQ ID NO:43) and Z:GCTTCAGCAGGACTTACACGGATGGCCCTG. (SEQ ID NO:44)Antibody Production and Western Blotting

The cDNA encoding SEBF was fused to the glutathione S-transferase (GST)gene in plasmid pGEX4T1™ (Amersham-Pharmacia Biotech). The fusionprotein (GST-SEBF) was expressed in BL21 cells. After lysis of the cellsthe fusion protein was purified on a glutathione column (Smith andJohnson, 1988). The GST tag was cleaved with thrombin(Amersham-Pharmacia Biotech) and removed from the extract by a secondpass on the glutathione column. Rabbit immunization was done accordingto Harlow and Lane (1988). Briefly, 50 μg of recombinant SEBF was usedto immunize rabbits followed by a second injection of 150 μg 30 dayslater to boost the immunological reaction. The rabbits were sacrificedfourteen days following the second injection. For western blot studiesthe SEBF antisera was used at a dilution 1:5000 and the antibody-antigeninteraction was revealed with the ECL™ detection kit (Amersham ParmaciaBiotech) according to manufacturers instructions.

Yeast Two-hybrid Screening

The two-hybrid screening was done using the DupLEX-A™ yeast two hybridsystem (OriGene, Rockville, Md., USA). The coding sequence for matureSEBF was inserted in frame with the LEX-A DNA binding domain in the baitvector pEG202. A tomato cDNA library made in the target vector pJG4–5was obtained from B. Baker at the U.S. Department of Agriculture (Zhanget al., 1999). Five million clones were screened in strain EGY48according to the manufacturers instructions. Each positive clone wasretransformed to verify the authenticity of the interaction. Thepositive clones were then sequenced on both strands using the T7sequencing kit (Amersham-Pharmacia Biotech). Translated proteins wereidentified through the BLASTX™ program at NCBI™.

c) Results

Identification of SEBF

Previous studies identified a negative regulatory sequence (SE) in thepromoter of the potato PR-10a gene (Matton et al., 1993; Després et al.,1995). We therefore sought to identify a nuclear protein able toregulate PR-10a expression through binding to this sequence. SincePR-10a appears to be positively regulated at the ERE by asingle-stranded DNA (ssDNA) binding protein (Desveaux et al., 2000), wesearched for proteins that could bind either single- or double-strandedforms of the SE. A crude nuclear extract of potato tubers was incubatedwith single- or double-stranded radiolabeled SE and analyzed byelectrophoretic mobility shift assay (EMSA). FIG. 1 demonstrates that anuclear factor, called SEBF for SE binding factor, bound the codingstrand of the SE (lane 1). SEBF was not able to bind the non-codingstrand (lane 3) or double-stranded DNA (lanes 4 through 6). Theseresults show that SEBF is a ssDNA binding protein that recognizes onlyone strand of the silencing element.

We have previously demonstrated that another cis-element of the PR-10apromoter also interacts with a ssDNA binding protein. This element,located between −135 and −105, interacts with the ssDNA binding factorPBF-2 (Després et al., 1995; Desveaux et al., 2000), which has beenshown to activate the transcription of a PR-10a-uidA reporter geneconstruct in protoplasts (Desveaux and Brisson, in preparation).Therefore, our data suggest that a large region of the PR-10a promoteris contacted by ssDNA binding proteins. A similar situation has beenreported for several genes in animals (reviewed by Rothman-Denes et al.,1998) and is illustrated by the promoter of the c-myc proto-oncogene,where several classes of single-stranded cis elements occur in vivo andare bound by various specific ssDNA binding factors (Michelotti et al.,1996).

Binding of SEBF to the SE Correlates with the Transcriptional Repressionof PR-10a

A mutational analysis of the SE was performed to determine whichnucleotides compose the SEBF binding site and whether these nucleotidesare important in vivo for the regulation of PR-10a expression. Mutatedforms of the SE coding strand were synthesized (FIG. 2A) and used asprobes in EMSA studies (FIG. 2B). The same mutations were alsointroduced into the PR-10a promoter fused to the uidA reporter geneencoding β-glucuronidase (GUS) and the resulting transcriptionalactivity was measured in a transient expression system (FIG. 2D). Asshown in FIG. 2B, binding of SEBF to mutants m3 (SEQ ID NO:3) and m4(SEQ ID NO:4) was significantly reduced compared to WT (SEQ ID NO:45).In contrast, introduction of these mutations in the promoter of PR-10aled to an increased activity of the reporter gene in transient studies(FIG. 2D, m3 and m4). Mutations that showed either a slight increase(FIG. 2B, m2 (SEQ ID NO:2)) or decrease (FIG. 2B, m5 (SEQ ID NO:5)) inbinding did not show altered transcriptional activities (FIG. 2D, m2 andm5). Binding of SEBF was completely abolished using mutant m1 (SEQ IDNO:1), where 80 percent of the sequence has been changed (FIG. 2B, m1).This absence of binding correlated with the highest transcriptionalactivity observed in transient studies (FIG. 2D, m1). Therefore, thetranscriptional activity of the PR-10a promoter is inversely correlatedto the in vitro binding of SEBF at the SE. This suggests that SEBF isthe trans-acting factor responsible for SE-mediated repression ofPR-10a.

The Sequence BTGTCNC Defines the DNA Binding Site of SEBF

Results presented in FIG. 2 indicate that the SEBF binding site islocated within the sequence GACTGTCAC (SEQ ID NO:26). To furtherdelineate this element, additional mutations were introduced into thisregion and their effect on SEBF binding was monitored by EMSA. Asindicated in FIG. 3, mutations affecting the sequence CTGTCAC (SEQ IDNO:25) dramatically reduced the binding of SEBF (FIGS. 3B and 3C,mutants m8 (SEQ ID NO:8), m9 (SEQ ID NO:8), m11 (SEQ ID NO:11), m13 (SEQID NO:13), m14 (SEQ ID NO:14), m15 (SEQ ID NO:15), m16 (SEQ ID NO:16),m17 (SEQ ID NO:17), m18 (SEQ ID NO:18)), whereas mutations outside thisregion did not affect binding (FIGS. 3B and 3C mutants m6 (SEQ ID NO:6),m7 (SEQ ID NO:7), m12 (SEQ ID NO:12), m20 (SEQ ID NO:20)). Mutations ofthe only A in this sequence did not affect binding (FIGS. 3B and 3C;mutants m12 (SEQ ID NO:12) and m19 (SEQ ID NO:19)). In fact, anynucleotides could substitute the A without affecting SEBF binding (notshown). Further analysis of the SEBF binding site revealed that thefirst C could be substituted by G or T. The only nucleotide that reducesSEBF binding at that specific position is A (FIG. 3, m13 (SEQ IDNO:13)). Therefore, the consensus SEBF binding site is BTGTCNC (SEQ IDNO:23), preferably YTGTCNC (SEQ ID NO:24), where N is all nucleotides, Bis C, G or T, and Y is C or T.

The SEBF Binding Bite is Present in Other Defense Genes

Table 5 present the results of a DNA database searches reveal that theSEBF binding site is present in the promoter of a number of genes knownto be induced by pathogens. These include: glucanases from tomato andrice (GenBank™ acc. Nos AF077340 and X58877); chitinases fromArabidopsis, bermudagrass, potato, rice and tobacco (GenBank™ acc. NosY14590, AF105426, AF153195, AF013581, X16938); PR-1 from barley andtobacco (GenBank™ acc. Nos Z48728 and X66942) and PR-10 from apple,birch, parsley and pea (GenBank™ acc. Nos AF020542, AJ289771, U48862 andU31669).

A previously identified repressor element of the CHN50 gene contains theSEBF binding site. This element was tested in EMSA to verify if it couldbe recognized by SEBF. FIGS. 9 and 12A demonstrate that SEBF can bindthe repressor element of the CHN50 gene suggesting a role for SEBF inthe regulation of this other defense genes. In addition, FIG. 12Bdemonstrates that the residues important for SEBF binding greatlycontribute to the repression of the CHN50 gene. Furthermore, a mutationthat abolishes SEBF binding (M2, FIG. 12A) results in derepression ofthe CHN50 gene (M2, FIG. 12B). Table 5 also shows that SEBF binding siteis found in numerous wound or stress inducible genes. Altogether theseresults suggests an important role for SEBF and its element SEBF inplant defense responses and more particularly in the regulation of plantdefense genes.

The SEBF Binding Site is Similar to AuxRE

As shown in Table 5, comparison of the SEBF binding site (BTGTCNC) withother regulatory elements reveals a strong similarity to the auxinresponse element (TGTCTC; SEQ ID NO:55) present in composite AuxRE.Analysis of the nucleotide 5′ to the TGTCTC of published AuxRE revealedan enrichment in purines (C or T) suggesting that most AuxRE are likelyto be bound by SEBF. Furthermore, AuxRE were shown to repress the actionof a constitutive enhancer element in the absence of auxins (Ulmasov etal., 1995) and they appear to be occupied, regardless of the auxinstatus, by a yet unidentified factor that can interact with ARF factors(Ulmasov et al., 1999). SEBF most probably corresponds to this factorsince: 1) it is present in the nucleus of leaves and tubers; 2) itfunctions as a transcriptional repressor (see below); and 3) it shouldbind to most Aux-RE, as exemplified by SEBF binding to one of the mostcharacterized AuxRE which is the D4 element of the GH3 gene of soybean(FIG. 11; Ulmasov et al., 1995).

Auxin is also known to negatively regulate several defense genes(Grosset et al., 1989; Jouanneau et al., 1991). Recently, an Arabidopsispleiotropic mutant was isolated which shows both increasedsusceptibility to pathogens and auxin insensitivity, demonstratingcomplex interactions between the pathways leading to these phenotypes(Mayda et al., 2000). These results, and the strong similarity betweenthe SEBF binding site and the AuxRE, raise the possibility that SEBFcould also be involved in hormonal control of gene expression.

Purification of SEBF

A cDNA for SEBF was cloned using information deduced from the amino acidsequence of the purified protein. SEBF was purified from tuber nucleiusing a combination of anion exchange chromatography and affinitypurification. Table 4 hereinbelow shows that a 20,700 fold purificationof SEBF was achieved.

TABLE 4 Purification of SEBF from potato tubers Total Total SpecificProtein Activity^(a) Activity Purifica- Yield Fraction (mg) (pg DNA) (pgDNA/mg) tion (fold) (%) Crude 112 3.3 × 10⁶ 2.9 × 10⁴ 1 100 nuclearQ-Seph^(b) 1.15 3.0 × 10⁶ 2.6 × 10⁶ 90 91 Aff.1^(c) 4.0 × 10^(−3d) 2.3 ×10⁶ 5.8 × 10⁸ 20,000 70 Aff.2^(e) 3.5 × 10^(−3d) 2.1 × 10⁶ 6.0 × 10⁸20,700 64 ^(a)Total activity determined by measuring labeled probe boundby SEBF in EMSA and calculating total pg of DNA bound in each fraction^(b)Q-Seph., Q-Sepharose Fast-Flow ™ anion exchange chromatography^(c)Aff.1, First round of DNA affinity chromatography ^(d)Estimated fromstained gels ^(e)Aff.2, Second round of DNA affinity chromatography

The analysis of the chromatographic fractions by SDS-PAGE and Coomassiestaining revealed two distinct bands of 29 and 28 kD in the mostpurified fraction (FIG. 4, lane 4). To determine which of these twobands possessed DNA-binding activity, proteins from the second affinitypurification (Aff.2) were transferred to nitrocellulose and probed withthe wild type SE oligonucleotide. Results indicate that the two purifiedproteins can interact independently with the silencing element (FIG. 4,lane 5). Both of these proteins were subjected to N-terminal sequenceanalysis.

Cloning of SEBF

A 40 amino acid sequence was obtained from N-terminal sequencing of the29 kD protein. Amino-terminal sequencing of the 28 kD protein showedmore than one amino acid at each cycle. However, a clear sequencesimilarity with that of the 29 kD protein could be, observed, suggestingthat the 28 kD band contained degraded forms of the 29 kD protein. Usingthe amino acid sequence obtained from the 29 kD protein, a PCR strategywas elaborated to clone a cDNA (see materials and methods). The sequenceof SEBF cDNA (SEQ ID NO:21) is shown in FIG. 10 whereas its amino acidsequence (SEQ ID NO:22), derived from the cDNA clone, is presented inFIG. 10 and in FIG. 5. The 40 residues determined by N-terminalsequencing (shown in bold in FIG. 5) are preceded by a 59 amino acidsequence with the attributes of a transit peptide. An acidic regionpresent in the N-terminal region of the mature protein precedes twoconsensus sequence type RNA-binding domains (cs-RBD, underlined in FIG.5). These domains, also known as RNA recognition motifs (RRM), RNPconsensus sequences (RNP-CS) or RNP motifs (Burd and Dreyfuss, 1994),are separated by a glycine rich region. As mention previously, databasesearches indicated a high degree of sequence similarity with a family ofnuclear encoded chloroplast RNA binding proteins (see Tables 1 to 3hereinbefore).

The presence of RNA binding domains in SEBF, and the multiple functionsassociated with this domain, suggests that SEBF could also play a rolein RNA processing. Tobacco chloroplast homologues of SEBF have beenshown to bind mRNA and intron-containing pre-tRNAs (Nakamura et al.,1999). They have also been shown to confer stability and ribonucleaseprotection to mRNA in the chloroplasts (Nakamura et al., 2001). Inaddition, RNA editing in chloroplasts, like mammalian nuclear editing,also involves the presence of hnRNPs (Lau et al., 1997; Hirose andSugiura, 2001). Therefore, SEBF represents a single-stranded-DNA bindingfactor that could be involved in the regulation of transcription ofnuclear genes and the control of gene expression in plastids by bindingto RNA.

To confirm that the cDNA-encoded protein functionally corresponds toSEBF, the protein was expressed as a GST fusion in E. coli, purified,and tested for binding to the SE. FIG. 2C shows that the recombinantprotein binds to SE oligonucleotides with binding specificity similar tonative SEBF (FIG. 2B).

Cellular Distribution of SEBF

The purification of SEBF from isolated nuclei (Table 4) indicates thatSEBF is a nuclear protein. However, the presence of a putative transitsequence in recombinant SEBF suggests that the protein could also bepresent in plastids. This led us to investigate the subcellularlocalization of SEBF. Antibodies were raised against recombinant SEBFand subcellular fractions were obtained from potato leaves. Theintegrity of each fraction was verified by enzymatic assays. FIG. 6shows that, as expected, alcohol dehydrogenase activity was restrictedto the cytosolic fraction. The nuclei were free of the chloroplasticmarker chlorophyll, and little contamination was observed in thecytosolic fraction. No detectable alkaline pyrophosphatase or nitritereductase activities were found in nuclear preparation. These twoenzymes are known to be located exclusively in the plastids (Miflin,1974; Gross and ap Rees, 1986). Western blot analysis performed on thesefractions revealed that a protein immunologically related to SEBF waspresent in chloroplasts and nuclei (FIG. 6). A single 29 kD band,corresponding to the molecular weight of the mature form of SEBF (FIG.6, Recombinant SEBF), is present in both cellular compartments. Noproteins of higher molecular weight were detected in any fraction,suggesting that if SEBF is synthesized as a precursor, it is efficientlyprocessed by peptidases prior to nuclear import.

Dual Localization of SEBF

The presence of a similar sized, immunologically-related protein to SEBFin chloroplasts suggests that this protein is efficiently targeted tothis cellular compartment. This is supported by the presence of aputative transit peptide encoded in the cDNA of SEBF and by studies inother species which have shown that SEBF homologues are present in thechloroplasts (Ye et al., 1991; Mieszczak et al., 1992; Ohta et al.,1995).

SEBF is a Single Copy Gene

To rule out the possibility that nuclear and chloroplastic SEBF could bethe product of different genes, genomic DNA was digested withrestriction enzymes and used for Southern blot analysis. As shown inFIG. 7A, probing the genomic DNA with an SEBF cDNA fragment resulted ina single EcoRI band and two HinDIII bands. These were the expectedresults for a single copy gene since no EcoRI sites and a single HinDIIIsite are found within the probe. The presence of two XbaI fragments isdue to the presence of an XbaI site in the third intron of the gene (notshown). Analysis of potato genomic DNA by PCR led to the identificationof three introns in the gene coding for SEBF (FIG. 7B). The introns arepositioned between nucleotides 454 and 455, between nucleotides 556 and557, and between nucleotides 869 and 870 of SEQ ID:21 for introns 1, 2and 3 respectively. These three introns are present at the same positionin homologues of SEBF in Arabidopsis and tobacco (Ye et al., 1991; Ohtaet al., 1995). These results clearly indicate that there is only onecopy of the SEBF gene in the potato genome. Since no introns are presentin the 5′ end of the gene (FIG. 7C), the absence of a transit peptide onthe purified protein is not due to alternative splicing. This wasconfirmed by sequencing 5′RACE products which all showed sequencesidentical to that of SEBF (not shown). These results are also supportedby the conservation of the genomic organization of this gene in tobaccoand Arabidopsis.

Taken together, the results indicate that the nuclear-localized SEBF isencoded by a single-copy gene and that the transit peptide is noteliminated by differential splicing. This suggests that both the nuclearSEBF and the chloroplast protein are derived from the same precursor. Asonly the mature form of SEBF is detected in the nucleus, this alsosuggests that the precursor can be processed outside the chloroplast.Similar results were found in potato for the enzyme starchphosphorylase, which is synthesized as a precursor and targeted to theamyloplast in young tubers but accumulates in a processed form in thecytoplasm of older tubers (Brisson et al., 1989). In line with thesedata, Dahlin and Cline (1991) reported that import of proteins intoplastids is developmentally regulated, with the plastids losing theirability to import precursors during maturation. This differential importof proteins could be controlled by phosphorylation of transit peptides,which has been shown to reduce chloroplast import rates (Waegemann andSoll, 1996). It will be interesting in the future to determine whetherthe subcellular localization of SEBF changes during development.Nonetheless, the dual localization of SEBF in chloroplasts and thenucleus makes this protein an ideal candidate for regulating metabolismin both subcellular compartments during the defense response. It is wellestablished that, in addition to the activation of nuclear defensegenes, infection leads to major changes in primary metabolism of theplant, including reductions in the rate of photosynthesis and synthesisof RuBisCO (Tang et al., 1996; Somssich and Hahlbrock, 1998). SEBF couldtherefore play a role in coordinating defense-induced changes in bothcompartments.

SEBF can Repress Transcription

The correlation between binding of SEBF to mutated forms of the SE andtheir activity as negative regulatory elements in protoplasts suggeststhat SEBF is the factor mediating SE dependent repression. This wasconfirmed by co-expressing SEBF and various reporter constructs inpotato protoplasts. Although repression was observed using the wild typepromoter construct, reporter activity was too low and the results werenot statistically significant (FIG. 8, WT). This result was expected aspromoter activity with the wild-type SE is already fully repressed. Tocircumvent this problem, we overexpressed SEBF in conjunction with aconstruction carrying the partially derepressed mutant m4 (see FIG. 2D),which is bound with less affinity than WT by recombinant and purifiedSEBF (FIGS. 2B an 2C). We expected that overexpression of SEBF wouldcompensate for the lower binding affinity to this mutant. FIG. 8demonstrates that indeed, a 68 percent decrease of promoter activity wasattained when SEBF was overexpressed, compared to the activity observedwith the overexpression of FKBP, a control protein (FIG. 8, m4).Repression was not seen when SEBF was overexpressed with mutant m1 (FIG.8, m1), to which it does not bind, confirming that the repressionobserved requires a cis-element bound by SEBF and is not due totethering of an activator or translational blocking. These resultsdemonstrate that SEBF participates in SE mediated PR-10a repression andthat expression of the SEBF precursor results in nuclear localizedactivity.

hnRNP are among the best characterized ssDNA binding proteins playing arole in the regulation of gene expression in eukaryotes. For example,hnRNP D and its homologues have been shown to activate (Tay et al.,1992; Tolnay et al., 2000; Lau et al., 2000) or repress transcriptionfrom a variety of promoters (Kamada and Miwa, 1992; Smidt et al., 1995;Chen et al., 1998). Another cs-RBD containing protein, human hnRNP A1,has been shown to repress transcription of the thymidine kinase gene(Lau et al., 2000). It is therefore not surprising that SEBF, a plantprotein containing cs-RBDs, can act as a transcriptional repressor.Other plant proteins containing two cs-RBDs, such as Arabidopsis FMV-3b,carnation CEBP-1, and tobacco ACBF were also able to bind specificregulatory cis-elements (Didier and Klee, 1992; Maxson and Woodson,1996; Séguin et al., 1997) and therefore represent potentialtranscriptional regulators.

SEBF Interacts with Pti4

The yeast two-hybrid system was used to determine to which proteins SEBFinteracts. Of five interactors identified, two corresponded to SEBF,indicating that the protein is able to form multimers, one correspondedto a protein with similarity to a DEAD-box like RNA helicase (Itadani etal., 1994), and two interactors corresponding to the tomatotranscription factor Pti4 (Zhou et al., 1997). Pti4 is known to interactwith the Pto resistance gene which controls the defense response intomato against bacterial pathogens. These results suggest that SEBFcould be involved in the regulation of defense genes regulated by Pti4and Pto.

With the two hybrid screen, other interactors (presented in Table 6)were also identified. SEBF could contribute to the regulation of theseproteins and the processes involving these interactors.

TABLE 5 Occurrence of the SEBF binding site GenBank ™ Gene Organism acc.No. Sequence Defense genes PR-10a Potato M29041 CTGTCAC PR-10b PotatoM29042 CTGTCAC PR-10 (ypr10b) Birch AJ289770 CTGTCTC PR-10 (ypr10a)Birch AJ289771 CTGTCAC PR-10 Apple AF020542 CTGTCAC PR-10 Parsley U48862TTGTCTC Chitinase Tobacco X51599 ATGTCTC Chitinase Potato AF153195TTGTCAC Chitinase Bermuda grass AF105426 TTGTCTC Chitinase ArabidopsisY14590 TTGTCTC Glucanase Rice X58877 CTGTCAC Glucanase Tomato AF077340CTGTCAC PR-1 Tobacco X66942 TTGTCAC CTGTCAC PR-1 Barley Z48728 TTGTCACOsmotin (PR-5) Tobacco S68111 TTGTCAC Osmotin (PR-5) Tobacco D76437GTGTCAC Peroxidase Wheat X85230 CTGTCAC Alternative oxidase Voodoo lilyZ15117 CTGTCAC Antifungal protein Gastrodia elata AF334813 CTGTCTCEthylene production ACC synthase Tomato AF043122 GTGTCAC ACC synthaseMungbean AB018355 CTGTCAC ACC synthase Potato 3 Z27235 TTGTCAC Potato 2Z27234 ATGTCCC ATGTCTC Potato 1 Z27233 TTGTCCC (2x) TTGTCTC GTGTCACATGTCTC ACC oxidase Banana AF030411 TTGTCTC Cellulase ethylene SoybeanU34754 CTGTCTC induced Wound and stress induced myrosinase-associated B.napus AJ223307 CTGTCAC protein wun-1 Sweet potato X17554 CTGTCAC osr40g2(salt stress) Rice Y08987 CTGTCAC Metabolism rubisco small Maize U09743CTGTCAC rubisco small C. reinhardtii X04472 CTGTCAC Starch branchingMaize AF072724 CTGTCAC enzyme ADP-glucose Barley AJ239130 CTGTCACpyrophosphorylase ADP-glucose Potato X96771 CTGTCAC (2x)pyrophosphorylase Nitrate transporter Barley AF189727 CTGTCAC Sulphatetransporter Barley AF075270 CTGTCAC chlorophyll a/ Wheat X05823 CTGTCACb-binding protein Others CDPK Rice Y13658 TTGTCTC CTGTCAC LAP (flowersp.) Tomato Y08305 CTGTCAC MADS box protein Rice AF204063 CTGTCAC GEG(fruit sp.) Gerbera AJ006273 CTGTCAC hybrida

TABLE 6 Proteins identified by two hybrid screening InteractionNucleotide BLAST^(a) C.C.^(b) Protein BLAST^(a) C.C.^(b) Strength Pti4mRNA, tomato 3.00E−93 Pti4, tomato 5.00E−26 Strong Pti4 mRNA, tomato 0Pti4, tomato 4.00E−39 Strong Pti4 mRNA, tomato 1.00E−101 Pti4, tomato6.00E−25 Strong cp29A mRNA, tobacco 9.00E−72 cp29 A, tobacco 1.00E−33Medium cp33 mRNA, tobacco 5.00E−67 cp33, tobacco   2E−28 Strong B2 mRNA,carrot 3.00E−13 B2, carrot 4.00E−09 Medium B2 mRNA, carrot 2.00E−46 B2,carrot 1.00E−57 Strong B2 mRNA, carrot 5.00E−37 B2, carrot 3.00E−28Strong B2 mRNA, carrot 1.00E−25 B2, carrot 9.00E−31 Medium B2 mRNA,carrot 2.00E−37 B2, carrot 4.00E−29 Medium B2 mRNA, carrot 2.00E−15 B2,carrot 8.00E−17 Strong B2 mRNA, carrot 7.00E−49 B2, carrot 7.00E−52Medium B2 mRNA, carrot 4.00E−13 B2, carrot 3.00E−10 Medium B2 mRNA,carrot 9.00E−11 B2, carrot 2.00E−06 Strong B2 mRNA, carrot 1.00E−16 B2,carrot 2.00E−11 Medium None — B2-like 0.44 Weak None — B2-like 3.00E−09Weak None — B2-like 2.00E−28 Weak None — B2-like 4.00E−25 Medium None —B2-like 2.00E−15 Weak None — B2-like 2.00E−05 Medium DB10 mRNA, tobacco2.00E−14 RNA helicase, tobacco 1.00E−04 Strong DB10 mRNA, tobacco 0.73None Strong DB10 mRNA, tobacco 0 RNA helicase, tobacco 2.00E−90 StrongRZ-1 mRNA, tobacco 1.00E−12 RZ-1 RNA binding protein, 0.075 Strongtobacco ^(a)Results using the BLASTN ™ program for nucleotide BLAST ™and BlastX ™ for protein BLAST ™ on the non redundant (nr) GenBank ™database. ^(b)C.C: Certainty Coefficient shows the probability that thisparticular sequence is chosen randomly. ^(c)The strength of theinteraction was qualitatively evaluated by coloration on X-galcontaining media.

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While several embodiments of the invention have been described, it willbe understood that the present invention is capable of furthermodifications, and this application is intended to cover any variations,uses, or adaptations of the invention, following in general theprinciples of the invention and including such departures from thepresent disclosure as to come within knowledge or customary practice inthe art to which the invention pertains, and as may be applied to theessential features hereinbefore set forth and falling within the scopeof the invention or the limits of the appended claims.

1. A transformed or transfected cell comprising an isolated nucleic acidsequence encoding a protein having a SEBF (silencing element bindingfactor) function, wherein the nucleic acid sequence is selected from thegroup consisting of a nucleic acid sequence having SEQ ID NO: 21; and anucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22.2. A transformed or transfected cell comprising an isolated or purifiedprotein having a SEBF function, said protein comprising an amino acidsequence selected from the group consisting of the amino acid sequenceencoded by nucleotides 68 to 937 of SEQ ID NO: 21 and the amino acidsequence having SEQ ID NO:
 22. 3. A cloning or expression vector, eachcomprising a nucleic acid sequence encoding a protein having a SEBFfunction, said nucleic acid sequence is selected from the groupconsisting of a nucleic acid sequence having SEQ ID NO: 21; and anucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22.4. The cloning or expression vector of claim 3, wherein it furthercomprises an inducible or constitutive promoter.
 5. A transformed plantexhibiting an altered expression level or biological activity of aproteinic nuclear factor having a specific binding activity to BTGTCNC(SEQ ID NO: 23), wherein said transformed plant exhibits a modifiedresistance or tolerance to a pathogen when compared to a correspondinguntransformed plant, and said proteinic nuclear factor is a polypeptideencoded by a nucleic acid sequence selected from the group consisting ofa nucleic acid sequence having SEQ ID NO: 21; and a nucleic acidsequence encoding the amino acid sequence of SEQ ID NO:
 22. 6. Atransformed plant exhibiting an altered expression level or biologicalactivity of a proteinic nuclear factor having a specific bindingactivity to BTGTCNC (SEQ ID NO: 23), wherein said transformed plantexhibits a modified resistance or tolerance to a pathogen when comparedto a corresponding untransformed plant, and wherein said proteinicnuclear factor is a SEBF protein comprising the amino acid sequenceencoded by nucleotides 68 to 937 of SEQ ID NO: 21 and the amino acidsequence having SEQ ID NO: 22.